Long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the most effective and widely used methods for controlling malaria vectors. The recent reductions in malaria morbidity and mortality across Africa has been attributed to a scale-up of these interventions and to better access to diagnostic testing and artemisinin combination therapy (ACT) to treat malaria [1]. Most national malaria control programmes have prioritized universal coverage of LLINs to populations at risk [2]. Campaigns of IRS are particularly appropriate for rapid transmission control. Both approaches require good organization and receptive communities [3]. LLIN effectiveness relies on people regularly sleeping under their nets. IRS is sometimes challenged by the complex organization required and by user-fatigue sometimes associated with recurrent rounds of spraying [3].

The recent development of insecticide-treated wall lining technology [4] offers the prospect of a novel system of insecticide delivery, which is more residual than IRS and requires limited behavioural change. Interior walls can be lined with polymer sheeting (wall lining) or net wall hangings impregnated with insecticide. Using advances in binder technology, these tools can be prepared in a long-lasting format that allows the insecticide to diffuse to the surface in a controlled fashion, making them a long-lasting alternative to IRS. Pyrethroid-treated durable wall lining has been manufactured commercially using this technique and its use on interior wall surfaces has shown potential to improve user compliance and overcome the operational constraints associated with IRS [4, 5]. Durable wall lining has the potential to remain efficacious on home walls for three to four years. However, with the increasing problem of pyrethroid resistance on malaria vector control [6, 7], non-pyrethroid forms of durable lining which can be used against pyrethroid-resistant malaria vectors are urgently needed [7, 8]. Such materials could significantly reduce reliance on pyrethroids and enhance capacity to interrupt malaria transmission whilst living with pyrethroid resistance.

Organophosphates and carbamates, having a differing mode of action to pyrethroids, are potential alternative classes of insecticide, which could be used on wall linings [9]. These classes are effective against pyrethroid-resistant mosquitoes when used as IRS or wall linings [10, 11]. However, resistance to organophophates and carbamates due to insensitive acetylcholinesterase (ace-1 ^R ) has been reported in some pyrethroid-resistant malaria vector populations in West Africa [12–14]. Malaria vector control programmes, confronted by such multiple resistance, may be left with no option than resort to using these classes until new types of insecticide with novel modes of action are identified and made available.

The combining of non-pyrethroid IRS and pyrethroid LLIN has been recommended for resistance management and for improving control of insecticide-resistant malaria vectors [7]. This resistance management tactic relies on insect genotypes resistant to the insecticide in one intervention being killed by the insecticide in the other intervention provided they are not resistant to both insecticides [15]. Population genetics modelling indicates that combinations are less likely to provide this advantage when resistance to both insecticides is already present at detectable frequencies in the targeted vector population [8, 15]. However, reality is often more complex than the prediction of models. Some combinations may still improve personal protection or enhance kill through biochemical or behavioural interactions [7, 15]. With limited alternatives available for malaria control, empirical studies are needed to demonstrate whether improved vector control can be expected when non-pyrethroid IRS or wall linings are combined with pyrethroid LLINs against a multiple insecticide-resistant vector population.

In the current study, the efficacy of organophosphate-treated wall linings (WL) and net wall hangings (NWH) applied alone and in combination with LLINs was compared with pyrethroid-treated WL against an Anopheles gambiae population of Tiassalé, southern Côte d’Ivoire, which is resistant to the main classes of insecticide used in adult vector control [13]. Differential selection of insecticide-resistant genotypes was investigated to assess the potential for resistance management.

The aim of the study was to evaluate the efficacy and selection of resistance by p-methyl-treated WL when either applied alone or in combination with LLINs against an An. gambiae population in southern Côte d’Ivoire, which was resistant to pyrethroids and organophosphates [13]. The reported trial was part of a multicentre trial designed to demonstrate as a proof of concept whether non-pyrethroid wall liners could provide benefits for control when combined with LLINs against malaria vector populations with differing levels of insecticide resistance. It was also hoped to assess their potential for resistance management. In the trial of similar interventions conducted in Burkina Faso where vectors were also resistant to pyrethroids but largely susceptible to organophosphates, the p-methyl WL and NWH were far more effective, killing almost all mosquitoes (>95%) that entered the huts even without the addition of LLIN [23]. The lower mortality rates achieved with p-methyl-treated WL and NWH in the Côte d’Ivoire study (50-65%) can therefore be attributed to the high levels of phenotypic resistance to organophosphates. Despite the poorer levels of control relative to the Burkina Faso study, p-methyl WL and NWH, proved to be a better option against this multiple insecticide-resistant vector population than commercial pyrethroid WL, which killed only 30% of mosquitoes entering the huts.

High vector mortality and personal protection against biting mosquitoes are the desired outcomes of any vector control tool or combination of tools. LLINs are very efficacious in areas of full susceptibility to pyrethroids, where they can induce high mortality rates in mosquito populations and provide personal protection to net users [24]. Although the insecticidal efficacy of LLINs may be compromised when confronted with moderate to high pyrethroid resistance, LLINs can still be protective as shown in both the present Côte d’Ivoire and previous Burkina Faso studies [23] owing to the barrier effect of the net and the residual killing effect of the pyrethroid. Hence, LLINs can remain an important public health intervention even against malaria vector populations, which have moderate levels of resistance to pyrethroids [8]. Against vector populations which are resistant to pyrethroids but largely susceptible to the insecticide applied on the walls, the combining of pyrethroid LLINs with non-pyrethroid IRS has shown, in small scale hut trials, improved levels of mortality (mostly due to the wall treatment) and improved personal protection (due to the LLIN) [10, 25]. Under such circumstances the combination appears to restore mortality rates to levels comparable to that achieved with LLINs alone in areas where vectors are susceptible to pyrethroids [24–26]. In the present study, the combination failed to provide improved mortality over the LLIN alone against a multiple insecticide-resistant malaria vector population. This is a very disturbing finding considering the limited classes of insecticides currently available for malaria vector control. Until a class of insecticide with a novel mode of action is developed for vector control, malaria programmes faced with such multiple insecticide resistance may have no suitable alternatives to complement or provide a boost to failing LLINs. The study demonstrates the threats and challenges that the malaria vector control community will face if such resistance to multiple insecticides is left unchecked and continues to spread.

In other parts of West Africa, the ace-1 ^R gene has often been reported in pyrethroid-resistant An. gambiae populations at low frequencies [12, 23, 27, 28]. While heterozygotes for ace-1 ^R did show some selective advantage over homozygotes for susceptibility in the Burkina Faso study [23], the Côte d’Ivoire Tiassalé population had a far higher frequency of ace-1 ^R and the use of organophosphate WL clearly demonstrated the survival and selection of ace-1 ^R genotypes. A parallel mechanistic investigation on the Tiassalé population has demonstrated gene duplication at the ace-1 ^R locus [16]; the duplication may account for the dominance and survival advantage of ace-1 ^R genotypes and would also explain the departure from Hardy-Weinberg expectation and the surplus of heterozygotes. While the number of mosquitoes collected and analysed for ace-1 ^R in the second (combination) trial was not huge, there was no convincing evidence that ace-1 ^R heterozygotes or homozygotes were less likely to survive exposure to the combination relative to the single p-methyl interventions or that the combination would manage resistance. This, together with the quite high survival rates among mosquitoes that bore no ace-1 ^R alleles, suggests the presence of another mechanism, independent of ace-1 ^R , going undetected in survivors, which was partly responsible for organophosphate resistance. Recent studies showed improved mortality of An. gambiae from Tiassalé exposed to bendiocarb, pyrethroids and an organophosphates (fenitrothion) with different synergists, thus implicating enhanced P450s and esterases in the resistance to all three classes of insecticide [16, 17, 29]. An investigation of the genetic basis of resistance in the Tiassalé population has associated genes from the CYP6 subfamily with resistance to pyrethroids and carbamates [16]. It is important to identify the specific enzyme families, which in association with the ace-1 ^R mechanism, combine to increase resistance to p-methyl in this vector population.

While no large-scale community trial has been published on the combined effects of pyrethroid LLIN and organophosphate IRS compared to LLIN alone, two community randomized trials of LLIN and carbamate IRS have been published recently: one in Tanzania [30] and one in Benin [31]. Both were in areas of high-frequency pyrethroid resistance and low-frequency carbamate resistance. The Tanzanian trial showed an added effect of the combination over LLIN alone, and this result was therefore consistent with the outcome of the Burkina Faso experimental hut trial of LLIN and OP wall liners (and local susceptibility status). The contrasting findings from the two multicentre hut trials in Burkina Faso and Côte d’Ivoire illustrate the uncertainty of outcome when faced with resistance to multiple insecticides rather than single insecticides. From the outcome of the Côte d’Ivoire trial, there can be no doubt that selection of multiple insecticide resistance will only make it harder to control malaria.

P-methyl WL and NWH performed better than pyrethroid WL against multiple pyrethroid and organophosphate-resistant An. gambiae. Combining p-methyl WL and NWH with LLINs provided no improvement in mortality and personal protection compared to the LLIN alone. There was no evidence that the combination of pyrethroid LLIN and organophosphate WL would prevent the selection of either kdr or ace-1 ^R resistance when both are present at detectable or moderate frequencies. The study demonstrates the challenge that malaria vector control programmes are faced with when confronted with such high levels of phenotypic resistance to multiple insecticides. Strategies of insecticide deployment or rotation to delay the rapid spread of the ace-1 ^R gene in Africa and the further development of multiple insecticide-resistant vector populations are urgently required.