BackgroundMalaria control through use of long-lasting insecticidal nets (LN) is threatened by the selection of anopheline mosquitoes strongly resistant to pyrethroid insecticides. To sustain future effectiveness it is essential to identify and evaluate novel insecticides suitable for nets. Mixtures of two insecticides with contrasting mode of action have the potential to kill resistant vectors and restore transmission control provided the formulation can withstand regular washing over the net’s life span.MethodThe efficacy of a novel mixture LN, Interceptor® G2, that combines the pyrrole chlorfenapyr and pyrethroid alpha-cypermethrin was evaluated under controlled household conditions (experimental hut trial) and by laboratory bioassay against pyrethroid resistant An. gambiae in Benin before and after standardized washing. Comparison arms included standard alpha-cypermethrin LN, nets hand-treated with chlorfenapyr-only and untreated nets.ResultsThe chlorfenapyr-alphacypermethrin LN demonstrated improved efficacy and wash resistance compared to a standard alpha-cypermethrin LN against pyrethroid resistant mosquitoes (resistance ratio 207). In experimental hut trial alpha-cypermethrin LN killed only 20% (95% CI 15–26%) of host-seeking An. gambiae whilst mixture LN killed 71% (95% CI 65–77%). Mixture LN washed 20 times killed 65% (95% CI 58–71%), and thus intensive washing reduced efficacy by only 6% (95% CI 1.3–11%). The chlorfenapyr net killed 76% (95% CI 70–81%). Personal protection and blood feeding inhibition did not differ between mixture and pyrethroid LN; however, the mixture LN was 2.5 (95% CI: 2.1–3.1) times more protective than untreated nets. Standard WHO cone bioassays conducted during day time hours failed to anticipate field efficacy but overnight tunnel tests successfully predicted mixture LN and chlorfenapyr net efficacy in field trials.ConclusionInterceptor® G2 LN demonstrates the potential to control transmission and provide community protection over the normal lifespan of long lasting nets where standard pyrethroid LN show signs of failing due to resistance.

BackgroundThe rapid selection of pyrethroid resistance throughout sub-Saharan Africa is a serious threat to malaria vector control. Chlorfenapyr is a pyrrole insecticide which shows no cross resistance to insecticide classes normally used for vector control and is effective on mosquito nets under experimental hut conditions. Unlike neurotoxic insecticides, chlorfenapyr owes its toxicity to disruption of metabolic pathways in mitochondria that enable cellular respiration. A series of experiments explored whether standard World Health Organization (WHO) guidelines for evaluation of long-lasting insecticidal nets, developed through testing of pyrethroid insecticides, are suitable for evaluation of non-neurotoxic insecticides.MethodsThe efficacy of WHO recommended cone, cylinder and tunnel tests was compared for pyrethroids and chlorfenapyr. To establish bioassay exposure times predictive of insecticide-treated net (ITN) efficacy in experimental hut trials, standard three-minute bioassays of pyrethroid and chlorfenapyr ITNs were compared with longer exposures. Mosquito behaviour and response to chlorfenapyr ITN in bioassays conducted at night were compared to day and across a range of temperatures representative of highland and lowland transmission.ResultsStandard three-minute bioassay of chlorfenapyr produced extremely low levels of mortality compared to pyrethroids. Thirty-minute day-time bioassay produced mortality closer to hut efficacy of chlorfenapyr ITN but still fell short of the WHO threshold. Overnight tunnel test with chlorfenapyr produced 100% mortality and exceeded the WHO threshold of 80%. The endogenous circadian activity rhythm of anophelines results in inactivity by day and raised metabolism and flight activity by night. A model which explains improved toxicity of chlorfenapyr ITN when tested at night, and during the day at higher ambient temperature, is that activation of chlorfenapyr and disruption of respiratory pathways is enhanced when the insect is more metabolically and behaviourally active.ConclusionsTesting according to current WHO guidelines is not suitable for certain types of non-neurotoxic insecticide which, although highly effective in field trials, would be overlooked at the screening stage of evaluation through bioassay. Testing methods must be tailored to the characteristics and mode of action of each insecticide class. The WHO tunnel test on night-active anophelines is the most reliable bioassay for identifying the toxicity of novel insecticides.

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BackgroundNovel indoor residual spraying (IRS) and long-lasting insecticidal net (LLIN) products aimed at improving the control of pyrethroid-resistant malaria vectors have to be evaluated in Phase II semi-field experimental studies against highly pyrethroid-resistant mosquitoes. To better understand their performance it is necessary to fully characterize the species composition, resistance status and resistance mechanisms of the vector populations in the experimental hut sites.MethodsBioassays were performed to assess phenotypic insecticide resistance in the malaria vector population at a newly constructed experimental hut site in Cové, a rice growing area in southern Benin, being used for WHOPES Phase II evaluation of newly developed LLIN and IRS products. The efficacy of standard WHOPES-approved pyrethroid LLIN and IRS products was also assessed in the experimental huts. Diagnostic genotyping techniques and microarray studies were performed to investigate the genetic basis of pyrethroid resistance in the Cové Anopheles gambiae population.ResultsThe vector population at the Cové experimental hut site consisted of a mixture of Anopheles coluzzii and An. gambiae s.s. with the latter occurring at lower frequencies (23 %) and only in samples collected in the dry season. There was a high prevalence of resistance to pyrethroids and DDT (>90 % bioassay survival) with pyrethroid resistance intensity reaching 200-fold compared to the laboratory susceptible An. gambiae Kisumu strain. Standard WHOPES-approved pyrethroid IRS and LLIN products were ineffective in the experimental huts against this vector population (8–29 % mortality). The L1014F allele frequency was 89 %. CYP6P3, a cytochrome P450 validated as an efficient metabolizer of pyrethroids, was over-expressed.ConclusionCharacterizing pyrethroid resistance at Phase II field sites is crucial to the accurate interpretation of the performance of novel vector control products. The strong levels of pyrethroid resistance at the Cové experimental hut station make it a suitable site for Phase II experimental hut evaluations of novel vector control products, which aim for improved efficacy against pyrethroid-resistant malaria vectors to WHOPES standards. The resistance genes identified can be used as markers for further studies investigating the resistance management potential of novel mixture LLIN and IRS products tested at the site.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-015-0981-z) contains supplementary material, which is available to authorized users.