Trends in the selection of insecticide resistance in Anopheles gambiae s.l. mosquitoes in northwest Tanzania during a community randomized trial of longlasting insecticidal nets and indoor residual spraying.

Anopheles gambiae s.l. (Diptera: Culicidae) in Muleba, Tanzania has developed high levels of resistance to most insecticides currently advocated for malaria control. The kdr mutation has almost reached fixation in An. gambiae s.s. in Muleba. This change has the potential to jeopardize malaria control interventions carried out in the region. Trends in insecticide resistance were monitored in two intervention villages using World Health Organization (WHO) susceptibility test kits. Additional mechanisms contributing to observed phenotypic resistance were investigated using Centers for Disease Control (CDC) bottle bioassays with piperonylbutoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) synergists. Resistance genotyping for kdr and Ace-1 alleles was conducted using quantitative polymerase chain reaction (qPCR). In both study villages, high phenotypic resistance to several pyrethroids and DDT was observed, with mortality in the range of 12-23%. There was a sharp decrease in mortality in An. gambiae s.l. exposed to bendiocarb (carbamate) from 84% in November 2011 to 31% in December 2012 after two rounds of bendiocarb-based indoor residual spraying (IRS). Anopheles gambiae s.l. remained susceptible to pirimiphos-methyl (organophosphate). Bendiocarb-based IRS did not lead to the reversion of pyrethroid resistance. There was no evidence for selection for Ace-1 resistance alleles. The need to investigate the operational impact of the observed resistance selection on the effectiveness of longlasting insecticidal nets and IRS for malaria control is urgent.

Evaluation of indoor residual spraying with the pyrrole insecticide chlorfenapyr against pyrethroid-susceptible Anopheles arabiensis and pyrethroid-resistant Culex quinquefasciatus mosquitoes.

Chlorfenapyr is a pyrrole insecticide with a unique non-neurological mode of action. Laboratory bioassays of chlorfenapyr comparing the mortality of pyrethroid-susceptible and -resistant Anopheles gambiae s.s. and Culex quinquefasciatus mosquitoes indicated that operational cross-resistance is unlikely to occur (resistance ratio ranged between 0 and 2.1). Three trials of chlorfenapyr indoor residual spraying were undertaken in experimental huts in an area of rice irrigation in northern Tanzania that supports breeding of A. arabiensis. Daily mosquito collections were undertaken to assess product performance primarily in terms of mortality. In the second trial, 250mg/m(2) and 500mg/m(2) chlorfenapyr were tested for residual efficacy over 6 months. Both dosages killed 54% of C. quinquefasciatus, whilst for A. arabiensis 250mg/m(2) killed 48% compared with 41% for 500mg/m(2); mortality was as high at the end of the trial as at the beginning. In the third trial, 250mg/m(2) chlorfenapyr was compared with the pyrethroid alpha-cypermethrin dosed at 30mg/m(2). Chlorfenapyr performance was equivalent to the pyrethroid against A. arabiensis, with both insecticides killing 50% of mosquitoes. Chlorfenapyr killed a significantly higher proportion of pyrethroid-resistant C. quinquefasciatus (56%) compared with alpha-cypermethrin (17%). Chlorfenapyr has the potential to be an important addition to the limited arsenal of public health insecticides for indoor residual control of A. arabiensis and pyrethroid-resistant species of mosquito.

Behavioural and insecticidal effects of organophosphate-, carbamate- and pyrethroid-treated mosquito nets against African malaria vectors.

Three insecticides - the pyrethroid deltamethrin, the carbamate carbosulfan and the organophosphate chlorpyrifos-methyl - were tested on mosquito nets in experimental huts to determine their potential for introduction as malaria control measures. Their behavioural effects and efficacy were examined in Anopheles gambiae Giles s.s. (Diptera: Culicidae) and Anopheles funestus Giles s.s. in Muheza, Tanzania, and in Anopheles arabiensis Patton and Culex quinquefasciatus Say in Moshi, Tanzania. A standardized dosage of 25 mg/m(2) plus high dosages of carbosulfan (50 mg/m(2), 100 mg/m(2) and 200 mg/m(2)) and chlorpyrifos-methyl (100 mg/m(2)) were used to compare the three types of insecticide. At 25 mg/m(2), the rank order of the insecticides for insecticide-induced mortality in wild An. gambiae and An. funestus was, respectively, carbosulfan (88%, 86%) > deltamethrin (79%, 78%) > chlorpyrifos-methyl (35%, 53%). The rank order of the insecticides for blood-feeding inhibition (reduction in the number of blood-fed mosquitoes compared with control) in wild An. gambiae and An. funestus was deltamethrin > chlorpyrifos-methyl > carbosulfan. Carbosulfan was particularly toxic to endophilic anophelines at 200 mg/m(2), killing 100% of An. gambiae and 98% of An. funestus that entered the huts. It was less effective against the more exophilic An. arabiensis (67% mortality) and carbamate-resistant Cx quinquefasciatus (36% mortality). Carbosulfan deterred anophelines from entering huts, but did not deter carbamate-resistant Cx quinquefasciatus. Deltamethrin reduced the proportion of insects engaged in blood-feeding, probably as a consequence of contact irritancy, whereas carbosulfan seemed to provide personal protection through deterred entry or perhaps a spatial repellent action. Any deployment of carbosulfan as an individual treatment on nets should be carried out on a large scale to reduce the risk of diverting mosquitoes to unprotected individuals. Chlorpyrifos-methyl was inferior to deltamethrin in terms of mortality and blood-feeding inhibition and would be better deployed on a net in combination with a pyrethroid to control insecticide-resistant mosquitoes.

Mosquitoes and bednets: testing the spatial positioning of insecticide on nets and the rationale behind combination insecticide treatments.

The recent development of pyrethroid resistance of operational significance in Anopheles gambiae s.l. is a major threat to the control of malaria in West Africa. The so-called '2-in-1' bednet, in which the top of the net is treated with a non-pyrethroid insecticide and the sides with pyrethroid, has been proposed as a way of maintaining efficacy in the wake of such resistance. A host-seeking female Anopheles mosquito must contact both the top and sides of a '2-in-1' net, however, for such nets to be useful in resistance management. In the present study, the interaction between mosquitoes and insecticide-treated bednets (ITN) was explored by restricting the insecticide to particular surfaces of the nets (top only or sides only) and then testing these nets, untreated nets and nets treated on all their surfaces in experimental huts, under simulated field conditions. Over the 6-week trial, there was no significant difference in An. arabiensis mortality between nets treated with pyrethroid on the top only (39.2%), sides only (39.6%) and all surfaces (39.7%), thus indicating that a female An. arabiensis usually contacts both the top and sides of a bednet during its host-seeking behaviour. The data on blood-feeding indicated, however, that the insecticide used on the sides of the net may be more important in preventing mosquito biting than that on the top. These results support the rationale behind the '2-in-1' nets. Such nets may have advantages over the use of nets treated on all surfaces with a mixture of insecticides that includes a non-pyrethroid component. With the '2-in-1', the more toxic component can be deployed on the top of the net, away from human contact, while the more repellent pyrethroid can be restricted to the sides, to prevent blood-feeding. With the scaling-up of ITN coverage and the need to preserve pyrethroid efficacy, more consideration should be given to switching from pyrethroid-only nets to 'combination' nets that have been treated with a pyrethroid and another insecticide. Since the mosquitoes that act as malarial vectors may contact all surfaces of a bednet during their host-seeking, spatial heterogeneity in insecticide levels over the surface of a net may not reduce that net's overall efficacy. Nets with a rather uneven distribution of insecticide (such as those that might be produced using home-treatment insecticide kits) may therefore be no less effective, prior to washing, than nets with a more even distribution of insecticide (such as long-lasting insecticidal nets produced under factory conditions).

Comparative efficacies of permethrin-, deltamethrin- and alpha-cypermethrin-treated nets, against Anopheles arabiensis and Culex quinquefasciatus in northern Tanzania.

Mosquito nets treated with permethrin, deltamethrin or alpha-cypermethrin at 25 mg/m(2) were evaluated in experimental huts in an area of rice irrigation near Moshi, in northern Tanzania. The nets were deliberately holed to resemble worn nets. The nets treated with permethrin offered the highest personal protection against Anopheles arabiensis (61.6% reduction in fed mosquitoes) and Culex quinquefasciatus (25.0%). Deltamethrin and alpha-cypermethrin provided lower personal protection against An. arabiensis (46.4% and 45.6%, respectively) and no such protection against Cx. quinquefasciatus. Permethrin performed poorly in terms of mosquito mortality, however, killing only 15.2% of the An. arabiensis and 9.2% of the Cx. quinquefasciatus exposed to the nets treated with this pyrethroid (after correcting for control mortality). The alpha-cypermethrin and deltamethrin performed marginally better, with respective mortalities of 32.8% and 33.0% for An. arabiensis and 19.4% and 18.9% for Cx quinquefasciatus. The poor killing effect of permethrin was confirmed in a second trial where a commercial, long-lasting insecticidal net based on this pyrethroid (Olyset) produced low mortalities in both An. arabiensis (11.8%) and Cx. quinquefasciatus (3.6%). Anopheles arabiensis survivors collected from the verandahs of the experimental huts and tested on 0.75%-permethrin and 0.05%-deltamethrin papers, in World Health Organization susceptibility kits, showed mortalities of 96% and 100%, respectively. The continued use of permethrin-treated nets is recommended for personal protection against An. arabiensis. In control programmes that aim to interrupt transmission of pathogens by mosquitoes and/or manage pyrethroid resistance in such vectors, a combination of a pyrethroid and another insecticide with greater killing effect should be considered.

Experimental hut evaluation of the pyrrole insecticide chlorfenapyr on bed nets for the control of Anopheles arabiensis and Culex quinquefasciatus.

OBJECTIVE: To determine the efficacy of chlorfenapyr against Anopheles arabiensis and Culex quinquefasciatus in East Africa and to identify effective dosages for net treatment in comparison with the commonly used pyrethroid deltamethrin. METHODS: Chlorfenapyr was evaluated on bed nets in experimental huts against A. arabiensis and C. quinquefasciatus in Northern Tanzania, at application rates of 100-500 mg/m(2). RESULTS: In experimental huts, mortality rates in A. arabiensis were high (46.0-63.9%) for all dosages of chlorfenapyr and were similar to that of deltamethrin-treated nets. Mortality rates in C. quinquefasciatus were higher for chlorfenapyr than for deltamethrin. Despite a reputation for being slow acting, >90% of insecticide-induced mortality in laboratory tunnel tests and experimental huts occurred within 24 h, and the speed of killing was no slower than for deltamethrin-treated nets. CONCLUSIONS: Chlorfenapyr induced low irritability and knockdown, which explains the relatively small reduction in blood-feeding rate. Combining chlorfenapyr with a more excito-repellent pyrethroid on bed nets for improved personal protection, control of pyrethroid-resistant mosquitoes and pyrethroid resistance management would be advantageous.