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Background: Attractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action—disruption of ATP mediated energy transfer in mitochondria—it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission. Methods: In this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments. Results: Both the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut). Conclusion: These results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field conditions. [ABSTRACT FROM AUTHOR]

Background: West African and Ifakara experimental huts are used to evaluate indoor mosquito control interventions, including spatial repellents and insecticides. The two hut types differ in size and design, so a side-by-side comparison was performed to investigate the performance of indoor interventions in the two hut designs using standard entomological outcomes: relative indoor mosquito density (deterrence), exophily (induced exit), blood-feeding and mortality of mosquitoes. Methods: Metofluthrin mosquito coils (0.00625% and 0.0097%) and Olyset® Net vs control nets (untreated, deliberately holed net) were evaluated against pyrethroid-resistant Culex quinquefasciatus in Benin. Four experimental huts were used: two West African hut designs and two Ifakara hut designs. Treatments were rotated among the huts every four nights until each treatment was tested in each hut 52 times. Volunteers rotated between huts nightly. Results: The Ifakara huts caught a median of 37 Culex quinquefasciatus/night, while the West African huts captured a median of 8/night (rate ratio 3.37, 95% CI: 2.30-4.94, P < 0.0001) and this difference in mosquito entry was similar for Olyset® Net and more pronounced for spatial repellents. Exophily was greater in the Ifakara huts with > 4-fold higher mosquito exit relative to the West African huts (odds ratio 4.18, 95% CI: 3.18-5.51, P < 0.0001), regardless of treatment. While blood-feeding rates were significantly higher in the West African huts, mortality appeared significantly lower for all treatments. Conclusions: The Ifakara hut captured more Cx. quinquefasciatus that could more easily exit into windows and eave traps after failing to blood-feed, compared to the West African hut. The higher mortality rates recorded in the Ifakara huts could be attributable to the greater proportions of Culex mosquitoes exiting and probably dying from starvation, relative to the situation in the West African huts. [ABSTRACT FROM AUTHOR]

Background: Malaria control today is threatened by widespread insecticide resistance in vector populations. The World Health Organization (WHO) recommends the use of a mixture of unrelated insecticides for indoor residual spraying (IRS) and long-lasting insecticidal nets (LNs) or as a combination of interventions for improved vector control and insecticide resistance management. Studies investigating the efficacy of these different strategies are necessary. Methods: The efficacy of Interceptor G2 LN, a newly developed LN treated with a mixture of chlorfenapyr (a pyrrole) and alpha-cypermethrin (a pyrethroid), was compared to a combined chlorfenapyr IRS and Interceptor LN (a standard alpha-cypermethrin LN) intervention in experimental huts in Cove Southern Benin, against wild, free-flying, pyrethroid-resistant Anopheles gambiae s.l. A direct comparison was also made with a pyrethroid-only net (Interceptor LN) alone and chorfenapyr IRS alone. Results: WHO resistance bioassays performed during the trial demonstrated a pyrethroid resistance frequency of >90% in the wild An. gambiae s.l. from the Cove hut site. Mortality in the control (untreated net) hut was 5%. Mortality with Interceptor LN (24%) was lower than with chlorfenapyr IRS alone (59%, P < 0.001). The combined Interceptor LN and chlorfenapyr IRS intervention and the mixture net (Interceptor G2 LN) provided significantly higher mortality rates (73 and 76%, respectively) and these did not differ significantly between both treatments (P = 0.15). Interceptor LN induced 46% blood-feeding inhibition compared to the control untreated net, while chlorfenapyr IRS alone provided none. Both mixture/combination strategies also induced substantial levels of blood-feeding inhibition (38% with combined interventions and 30% with Interceptor G2 LN). A similar trend of improved mortality of pyrethroid-resistant An. gambiae s.l. from Cove was observed with Interceptor G2 LN (79%) compared to Interceptor LN (42%, P < 0.001) in WHO tunnel tests. Conclusion: The use of chlorfenapyr and alpha-cypermethrin together as a mixture on nets (Interceptor G2 LN) or a combined chlorfenapyr IRS and pyrethroid LN intervention provides improved control of pyrethroid-resistant malaria vectors by inducing significantly higher levels of mortality through the chlorfenapyr component and providing personal protection through the pyrethroid component. Both strategies are comparable in their potential to improve the control of malaria transmitted by pyrethroid resistant mosquito vectors. [ABSTRACT FROM AUTHOR]

Background: Novel 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. Methods: Bioassays 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. Results: The 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. Conclusion: Characterizing 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. [ABSTRACT FROM AUTHOR]

Background: Universal coverage with long-lasting insecticidal mosquito nets (LLIN) or indoor residual spraying (IRS) of houses remain the primary strategies for the control of mosquito vectors of malaria. Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed if malaria vector control is to remain effective. Indoxacarb is an oxadiazine insecticide that is effective as an oral and contact insecticide against a broad spectrum of agricultural pests and, due to its unique site of action, no cross-resistance has been detected through mechanisms associated with resistance to insecticides currently used in public health. Methods: WHO tunnel tests of host seeking mosquitoes were carried out as a forerunner to experimental hut trials, to provide information on dosage-dependent mortality, repellency, and blood-feeding inhibition. A dosage range of indoxacarb treated netting (100-1000 mg/m2) was tested against a pyrethroid susceptible strain of Anopheles gambiae. In addition, efficacy of indoxacarb 500 mg/m2 was compared with a standard pyrethroid formulation against pyrethroid susceptible and resistant Culex quinquefasciatus. Dosages between 25 and 300 mg/m2 indoxacarb were tested in tunnel tests and in ball-frame bioassays as mixtures with alphacypermethrin 25 mg/m2 and were compared with singly applied treatments against an insectary reared pyrethroid resistant strain of Cx. quinquefasciatus originally collected in Cotonou, Benin. Results: There was a dosage-dependent response in terms of indoxacarb induced mortality, with dosages >100 mg/ m2 producing the best mortality response. In tunnel tests indoxacarb 500 mg/m2 exceeded WHOPES thresholds with >80 % mortality of adult An. gambiae and blood-feeding inhibition of 75 %. No cross-resistance to indoxacarb was detected through mechanisms associated with resistance to pyrethroid insecticides and was equally effective against susceptible and resistant strains of Cx. quinquefasciatus. Indoxacarb 500 mg/m2 killed 75 % of pyrethroid resistant Cx. quinquefasciatus compared with only 21 % mortality with alphacypermethrin 40 mg/m2. Mixtures of indoxacarb with pyrethroid produced an additive response for both mortality and blood-feeding inhibition. The best performing mixture (indoxacarb 200 mg/m2 + alphacypermethrin 25 mg/m2) killed 83 % of pyrethroid resistant Cx. quinquefasciatus and reduced blood-feeding by 88 %, while alphacypermethrin only killed 36 % and inhibited blood-feeding by 50 %. Conclusions: New insecticides with different modes of action to those currently used in mosquito vector control are urgently needed. Indoxacarb shows great promise as a mixture with a pyrethroid and should be evaluated in experimental hut trials to determine performance against wild free-flying, pyrethroid resistant An. gambiae and washresistant formulations developed. [ABSTRACT FROM AUTHOR]

Background: Resistance of malaria vectors to pyrethroids threatens the effectiveness of long-lasting insecticidal nets (LLINs) as a tool for malaria control. Recent experimental hut and observational studies in Benin show that pyrethroid resistance reduces the insecticidal effect and personal protection of LLINs especially when they become torn. The World Health Organization has proposed a threshold for when nets are "too torn" at 1,000 cm² for rectangular holes and 790 cm² for round holes. This study examines whether there is a threshold above which LLINs no longer reduce malaria transmission. Methods: Intact and artificially-holed LLINs under three months old and untreated nets were tested by releasing mosquitoes from a susceptible Anopheles gambiae colony, a pyrethroid-resistant An. gambiae population and a resistant Culex quinquefasciatus population in closed experimental huts in Southern Benin, West Africa. The efficacy of LLINs and untreated nets was evaluated in terms of protection against blood feeding, insecticidal effect and potential effect on malaria transmission. Results: Personal protection by both LLINs and untreated nets decreased exponentially with increasing holed surface area, without evidence for a specific threshold beyond which LLINs could be considered as ineffective. The insecticidal effect of LLINs was lower in resistant mosquitoes than in susceptible mosquitoes, but holed surface area had little or no impact on the insecticidal effect of LLINs. LLINs with 22,500 cm² holed surface area and target insecticide content provided a personal protection of 0.60 (95 % CI 0.44-0.73) and a low insecticidal effect of 0.20 (95 % CI 0.12-0.30) against resistant An. gambiae. Nevertheless, mathematical models suggested that if 80 % of the population uses such nets, they could still prevent 94 % (95 % CI 89-97 %) of transmission by pyrethroid-resistant An. gambiae. Conclusions: Even though personal protection by LLINs against feeding mosquitoes is strongly reduced by holes, the insecticidal effect of LLINs is independent of the holed surface area, but strongly dependent on insecticide resistance. Badly torn nets that still contain insecticide have potential to reduce malaria transmission. The relationship between LLIN integrity and efficacy needs to be understood in order to guide LLIN distribution policy. [ABSTRACT FROM AUTHOR]

Background: The 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. Methods: The 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. Results: Standard 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. Conclusions: Testing 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. [ABSTRACT FROM AUTHOR]

Background Malaria control programmes across Africa and beyond are facing increasing insecticide resistance in the major anopheline vectors. In order to preserve or prolong the effectiveness of the main malaria vector interventions, up-to-date and easily accessible insecticide resistance data that are interpretable at operationally-relevant scales are critical. Herein we introduce and demonstrate the usefulness of an online mapping tool, IR Mapper. Methods A systematic search of published, peer-reviewed literature was performed and Anopheles insecticide susceptibility and resistance mechanisms data were extracted and added to a database after a two-level verification process. IR Mapper (www.irmapper.com) was developed using the ArcGIS for JavaScript Application Programming Interface and ArcGIS Online platform for exploration and projection of these data. Results Literature searches yielded a total of 4,084 susceptibility data points for 1,505 populations, and 2,097 resistance mechanisms data points for 1,000 populations of Anopheles spp. collected via recommended WHO methods from 54 countries between 1954 and 2012. For the Afrotropical region, data were most abundant for populations of An. gambiae, and pyrethroids and DDT were more commonly used in susceptibility assays (51.1 and 26.8% of all reports, respectively) than carbamates and organophosphates. Between 2001 and 2012, there was a clear increase in prevalence and distribution of confirmed resistance of An. gambiae s.l. to pyrethroids (from 41 to 87% of the mosquito populations tested) and DDT (from 64 to 91%) throughout the Afrotropical region. Metabolic resistance mechanisms were detected in western and eastern African populations and the two kdr mutations (L1014S and L1014F) were widespread. For An. funestus s.l., relatively few populations were tested, although in 2010-2012 resistance was reported in 50% of 10 populations tested. Maps are provided to illustrate the use of IR Mapper and the distribution of insecticide resistance in malaria vectors in Africa. Conclusions The increasing pyrethroid and DDT resistance in Anopheles in the Afrotropical region is alarming. Urgent attention should be afforded to testing An. funestus populations for metabolic resistance mechanisms. IR Mapper is a useful tool for investigating temporal and spatial trends in Anopheles resistance to support the pragmatic use of insecticidal interventions. [ABSTRACT FROM AUTHOR]

Background: In Benin, around four million Long-Lasting Insecticide-treated Nets were freely distributed to household to prevent malaria in 2011. In contrast to a previous campaign that targeted only children under 5 years and pregnant women, this distribution campaign was conducted in order to achieve universal coverage. This study presents the results of LLIN coverage and utilization after the distribution campaign. Methods: The study was a cross-sectional household survey which utilized a stratified two-stage cluster sampling design. The strata represented the twelve departments covered by the national distribution campaign in 2011 and included a total of 4,800 households randomly selected in the country. A questionnaire adapted from the standard Malaria Indicator Survey (MIS) Household Questionnaire was used. Data were entered using QPS software and analyzed with R 2.14.1. Results: LLIN ownership was 86.4% (74 - 94). On average, each household received 3 LLINs (2-4). The proportion of households that met the ratio one net for two persons was 77%. The proportions of individuals sleeping under LLINs were high (84.8%). LLIN use among urban residents was 10% lower than in effective users from rural areas (P = 0.00224). Conclusions: The universal distribution campaign conducted in Benin has increased LLIN ownership and use in the community. But additional efforts are need to improve and maintain LLIN coverage [ABSTRACT FROM AUTHOR]

Background: Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions. Methods: This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions. Results: The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5x for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%. Conclusions: This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate. [ABSTRACT FROM AUTHOR]

Background: Urban agricultural practices are expanding in several cities of the Republic of Benin. This study aims to assess the impact of such practices on transmission of the malaria parasite in major cities of Benin. Method: A cross sectional entomological study was carried out from January to December 2009 in two vegetable farming sites in southern Benin (Houeyiho and Acron) and one in the northern area (Azèrèkè). The study was based on sampling of mosquitoes by Human Landing Catches (HLC) in households close to the vegetable farms and in others located far from the farms. Results: During the year of study, 71,678 female mosquitoes were caught by HLC of which 25% (17,920/71,678) were Anopheles species. In the areas surveyed, the main malaria parasite, Plasmodium falciparum was transmitted in the south by Anopheles gambiae s.s. Transmission was high during the two rainy seasons (April to July and October to November) but declined in the two dry seasons (December to March and August to September). In the north, transmission occurred from June to October during the rainy season and was vehicled by two members of the An. gambiae complex: Anopheles gambiae s.s. (98%) and Anopheles arabiensis (2%). At Houeyiho, Acron and Azèrèkè, the Entomological Inoculation Rates (EIRs) and the Human Biting Rates (HBRs) were significantly higher during the dry season in Households Close to Vegetable Farms (HCVF) than in those located far from the vegetable areas (HFVF) (p < 0.05.). However, there were no significant differences in HBRs or EIRs between HCFV and HFVF during the rainy seasons at these sites (p > 0.05). The knock-down resistance (kdr) mutation was the main resistance mechanism detected at high frequency (0.86 to 0.91) in An. gambiae s.l. at all sites. The ace-1R mutation was also found but at a very low frequency (< 0.1). Conclusion: These findings showed that communities living close to vegetable farms are permanently exposed to malaria throughout the year, whereas the risk in those living far from such agricultural practices is limited and only critical during the rainy seasons. Measures must be taken by African governments to create awareness among farmers and ultimately decentralize farming activities from urban to rural areas where human-vector contact is limited. [ABSTRACT FROM AUTHOR]

Background: Mosquito-borne diseases are still a major health risk in many developing countries, and the emergence of multi-insecticide-resistant mosquitoes is threatening the future of vector control. Therefore, new tools that can manage resistant mosquitoes are required. Laboratory studies show that entomopathogenic fungi can kill insecticide-resistant malaria vectors but this needs to be verified in the field. Methods: The present study investigated whether these fungi will be effective at infecting, killing and/or modifying the behaviour of wild multi-insecticide-resistant West African mosquitoes. The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were separately applied to white polyester window netting and used in combination with either a permethrin-treated or untreated bednet in an experimental hut trial. Untreated nets were used because we wanted to test the effect of fungus alone and in combination with an insecticide to examine any potential additive or synergistic effects. Results: In total, 1125 female mosquitoes were collected during the hut trial, mainly Culex quinquefasciatus Say. Unfortunately, not enough wild Anopheles gambiae Giles were collected to allow the effect the fungi may have on this malaria vector to be analysed. None of the treatment combinations caused significantly increased mortality of Cx. quinquefasciatus when compared to the control hut. The only significant behaviour modification found was a reduction in blood feeding by Cx. quinquefasciatus, caused by the permethrin and B. bassiana treatments, although no additive effect was seen in the B. bassiana and permethrin combination treatment. Beauveria bassiana did not repel blood foraging mosquitoes either in the laboratory or field. Conclusions: This is the first time that an entomopathogenic fungus has been shown to reduce blood feeding of wild mosquitoes. This behaviour modification indicates that B. bassiana could potentially be a new mosquito control tool effective at reducing disease transmission, although further field work in areas with filariasis transmission should be carried out to verify this. In addition, work targeting malaria vector mosquitoes should be carried out to see if these mosquitoes manifest the same behaviour modification after infection with B. bassiana conidia. [ABSTRACT FROM AUTHOR]

Background: Scaling up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) with support from the Global Fund and President's Malaria Initiative is providing increased opportunities for malaria control in Africa. The most cost-effective and longest-lasting residual insecticide DDT is also the most environmentally persistent. Alternative residual insecticides exist, but are too short-lived or too expensive to sustain. Dow Agrosciences have developed a microencapsulated formulation (CS) of the organophosphate chlorpyrifos methyl as a cost-effective, long-lasting alternative to DDT. Methods: Chlorpyrifos methyl CS was tested as an IRS or ITN treatment in experimental huts in an area of Benin where Anopheles gambiae and Culex quinquefasiactus are resistant to pyrethroids, but susceptible to organophosphates. Efficacy and residual activity was compared to that of DDT and the pyrethroid lambdacyalothrin. Results: IRS with chlorpyrifos methyl killed 95% of An. gambiae that entered the hut as compared to 31% with lambdacyhalothrin and 50% with DDT. Control of Cx. quinquefasciatus showed a similar trend; although the level of mortality with chlorpyrifos methyl was lower (66%) it was still much higher than for DDT (14%) or pyrethroid (15%) treatments. Nets impregnated with lambdacyhalothrin were compromized by resistance, killing only 30% of An. gambiae and 8% of Cx. quinquefasciatus. Nets impregnated with chlorpyrifos methyl killed more (45% of An gambiae and 15% of Cx. quinquefasciatus), but its activity on netting was of short duration. Contact bioassays on the sprayed cement-sand walls over the nine months of monitoring showed no loss of activity of chlorpyrifos methyl, whereas lambdacyhalothrin and DDT lost activity within a few months of spraying. Conclusion: As an IRS treatment against pyrethroid resistant mosquitoes chlorpyrifos methyl CS outperformed DDT and lambdacyhalothrin. In IRS campaigns, chlorpyrifos methyl CS should show higher, more-sustained levels of malaria transmission control than conventional formulations of DDT or pyrethroids. The remarkable residual activity indicates that cost-effective alternatives to DDT are feasible through modern formulation technology. [ABSTRACT FROM AUTHOR]

Background: The emergence of pyrethroid resistance in Anopheles gambiae has become a serious concern to the future success of malaria control. In Benin, the National Malaria Control Programme has recently planned to scaling up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) for malaria prevention. It is, therefore, crucial to monitor the level and type of insecticide resistance in An. gambiae, particularly in southern Benin where reduced efficacy of insecticide-treated nets (ITNs) and IRS has previously been reported. Methods: The protocol was based on mosquito collection during both dry and rainy seasons across forty districts selected in southern Benin. Bioassay were performed on adults collected from the field to assess the susceptibility of malaria vectors to insecticide-impregnated papers (permethrin 0.75%, delthamethrin 0.05%, DDT 4%, and bendiocarb 0.1%) following WHOPES guidelines. The species within An. gambiae complex, molecular form and presence of kdr and ace-1 mutations were determined by PCR. Results: Strong resistance to permethrin and DDT was found in An. gambiae populations from southern Benin, except in Aglangandan where mosquitoes were fully susceptible (mortality 100%) to all insecticides tested. PCR showed the presence of two sub-species of An. gambiae, namely An. gambiae s.s, and Anopheles melas, with a predominance for An. gambiae s.s (98%). The molecular M form of An. gambiae was predominant in southern Benin (97%). The kdr mutation was detected in all districts at various frequency (1% to 95%) whereas the Ace-1 mutation was found at a very low frequency (⩽ 5%). Conclusion: This study showed a widespread resistance to permethrin in An. gambiae populations from southern Benin, with a significant increase of kdr frequency compared to what was observed previously in Benin. The low frequency of Ace-1 recorded in all populations is encouraging for the use of bendiocarb as an alternative insecticide to pyrethroids for IRS in Benin. [ABSTRACT FROM AUTHOR]

Background: Insecticide treated plastic sheeting (ITPS), sometimes known as durable lining, has potential as a longlasting insecticidal surface for malaria vector control when used as lining for interior walls and ceilings inside the home. Against a backdrop of increasing long lasting net (LN) coverage, we examined the effect of combining permethrin-treated plastic sheeting (ITPS) with LNs in Burkina Faso. Methods: A verandah trap experimental hut trial of ITPS with or without Olyset LN was conducted in the Vallée du Kou near Bobo-Dioulasso, where the two molecular forms of Anopheles gambiae s.s., S (frequency 65%) and M (frequency 35%), occur. The S form is mostly pyrethroid resistant (Fkdr = 92%) owing to the kdr mechanism, and the M form is mostly kdr susceptible (Fkdr = 7%). The treatment arms included ITPS, Olyset, ITPS plus Olyset, ITPS plus untreated net (with or without holes), and untreated control. Results: ITPS was significantly inferior to Olyset LN in terms of mortality (37% vs 63%), blood feeding inhibition (20% vs 81%) and deterrence (0 vs 42%) effects, and hence altogether inferior as a means of personal protection (16% vs 89%). The addition of ITPS to Olyset did not improve mortality (62%), blood feeding inhibition (75%), deterrence (50%) or personal protection (88%) over that of Olyset used alone. Use of untreated nets - both holed and intact - with ITPS provided greater protection from blood-feeding. The intact net/ITPS combination killed more mosquitoes than ITPS on its own. Conclusions: Although ITPS has a potential role for community control of malaria, at low coverage it is unlikely to be as good as Olyset LNs for household protection. The combination of pyrethroid IRS and pyrethroid LN - as practiced in some countries - is unlikely to be additive except, perhaps, at high levels of IRS coverage. A combination of LN and ITPS treated with an alternative insecticide is likely to be more effective, particularly in areas of pyrethroid resistance. [ABSTRACT FROM AUTHOR]

Introduction: Insecticide-treated nets (ITN) are an important method of preventing malaria. To remain effective, they need to be re-treated with pyrethroid insecticide at approximately yearly intervals. Systems for re-treating nets in Africa are limited, and the vast majority of nets in use have never been treated or were treated only once. Bayer Environmental Science (BES) has developed a long-lasting formulation 'KO-Tab 1-2-3 ®' which can be applied to the net post-manufacture, under field conditions, and renders the insecticide wash-resistant. Methods: The performance of polyester nets treated with three kinds of BES long-lasting formulations, a conventional ITN (treated with standard KO-Tab) and PermaNet 2.0 were evaluated after washing samples of treated netting up to 30 times using standard WHO procedures. Performance was measured using 'three-minute exposure' and 'median time to knockdown' bioassay tests and by measuring the levels of deltamethrin using high-pressure liquid chromatography. Results: The conventional ITN was largely stripped of deltamethrin within 5-10 washes and insecticidal efficacy in bioassay declined to suboptimal levels. With PermaNet and KO-Tab 1-2-3 the loss of deltamethrin was much slower: insecticide content halved within 20 washes and there was no loss of biological efficacy in three-minute exposure bioassays in WHO cylinders even after 30 washes. After 30 washes there remained on the netting 16% (4.4 mg/m ²) of the loading dose of KO-Tab 1-2-3 and 28% (18.8 mg/m ²) of the loading dose of PermaNet. Conclusion: KO-Tab 1-2-3 was confirmed to be a long-lasting insecticide formulation. This finding raises the prospect of conventional polyester nets being converted into long-lasting insecticidal nets through simple dipping in the community or at home. This single development, if widely adopted, could transform the malaria control landscape in Africa and have a major impact on malaria. [ABSTRACT FROM AUTHOR]

Background: Vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), have significantly contributed to malaria prevention efforts in sub-Saharan Africa. However, insecticide resistance has seriously hampered their efficacy in recent years and new tools are essential to further progress. In2Care® EaveTubes (ETs) are an inexpensive, new resistance-breaking vector control product under World Health Organization (WHO) evaluation informed by mosquito ecology to efficiently target malaria vectors. By installing ETs in the walls of the house at the eave level that funnel the natural airflow, mosquitoes are drawn in by the same heat and odor cues that typically attract them through open eaves. Once inside an ET, mosquitoes are exposed to insecticide-treated netting placed inside the ET. The aim of this study is to test whether ETs as stand-alone tool have an effect on the epidemiology of malaria in villages where houses have been modified with the ET intervention., Methods: A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of ETs on clinical malaria incidence in children living in Côte d'Ivoire. Thirty-four villages will be selected based on population size and the proportion of houses suitable for modification with ETs (17 treatment arms (ETs + LLINs, 17 control arms (LLINs only)). Based on the population census, 55 households per cluster with eligible children (i.e., between the ages of 6 months to 8 years old at the start of the study) will be randomly selected for recruitment into the active detection cohorts. In the treatment arm, we will enroll eligible children who reside in ET-treated houses. The intervention and control cohorts will be followed for 4 months for baseline covariate measurements and 24 months with intervention. During case detection visits, blood samples will be taken from all febrile children and tested for malaria infection with rapid diagnostic tests (RDTs). All positive clinical malaria infections will be treated. To estimate the impact of the ET on malaria vector densities, entomological measurements (indoor sampling with CDC traps) will be conducted monthly in 20 clusters (10 ET, 10 Control) in 10 randomly selected households per cluster. To estimate the infectiousness of malaria vectors, sporozoite rates will be measured in subsets of the collected mosquito samples., Discussion: Findings will serve as an efficacy trial of ETs and will be submitted to the WHO Vector Control Advisory Group (VCAG) for assessment of public health value. Entomological outcomes will also be measured as proxies of malaria transmission to help develop guidelines for the evaluation of future In2Care® ETs products., Trial Registration: ClinicalTrials.gov NCT05736679. Registered on 10 February 2023., (© 2023. The Author(s).)

Background: Insecticide-treated wall lining (ITWL) is a new concept in malaria vector control. Some Anopheles gambiae populations in West Africa have developed resistance to all the main classes of insecticides. It needs to be demonstrated whether vector control can be improved or resistance managed when non-pyrethroid ITWL is used alone or together with long-lasting insecticidal nets (LLINs) against multiple insecticide-resistant vector populations., Methods: Two experimental hut trials were carried out as proofs of concept to evaluate pirimiphos methyl (p-methyl)-treated plastic wall lining (WL) and net wall hangings (NWH) used alone and in combination with LLINs against multiple insecticide-resistant An. gambiae in Tiassalé, Côte d'Ivoire. Comparison was made to commercial deltamethrin WL and genotypes for kdr and ace-1R resistance were monitored., Results: The kdr and ace-1R allele frequencies were 0.83 and 0.44, respectively. Anopheles gambiae surviving discriminating concentrations of deltamethrin and p-methyl in WHO resistance tests were 57 and 96%, respectively. Mortality of free-flying An. gambiae in huts with p-methyl WL and NWH (66 and 50%, respectively) was higher than with pyrethroid WL (32%; P<0.001). Mortality with LLIN was 63%. Mortality with the combination of LLIN plus p-methyl NWH (61%) or LLIN plus p-methyl WL (73%) did not significantly improve upon the LLIN alone or p-methyl WL or NWH alone. Mosquitoes bearing the ace-1R were more likely to survive exposure to p-methyl WL and NWH. Selection of heterozygote and homozygote ace-1R or kdr genotypes was not less likely after exposure to combined LLIN and p-methyl treatments than to single p-methyl treatment. Blood-feeding rates were lower in huts with the pyrethroid LLIN (19%) than with p-methyl WL (72%) or NWH (76%); only LLIN contributed to personal protection., Conclusions: Combining p-methyl WL or NWH with LLINs provided no improvement in An. gambiae control or personal protection over LLIN alone in southern Côte d'Ivoire; neither did the combination manage resistance. Additional resistance mechanisms to kdr and ace-1R probably contributed to the survival of pyrethroid and organophophate-resistant mosquitoes. The study demonstrates the challenge that malaria control programmes will face if resistance to multiple insecticides continues to spread.

Background: The growing development of pyrethroid resistance constitutes a serious threat to malaria control programmes and if measures are not taken in time, resistance may compromise control efforts in the foreseeable future. Prior to Long Lasting Insecticidal Nets (LLINs) distribution in Eastern Cote d'Ivoire, we conducted bioassays to inform the National Malaria Control Programme of the resistance status of the main malaria vector, Anopheles gambiae s. s. and the need for close surveillance of resistance., Methods: Larvae of An. gambiae s. s. were collected in two areas of Adzopé (Port-Bouët and Tsassodji) and reared to adults. WHO susceptibility tests with impregnated filter papers were carried out to detect resistance to three pyrethroids commonly used to develop LLINs: permethrin 1%, deltamethrin 0.05% and lambda-cyhalothrin 0.05%. Molecular assays were conducted to detect M and S forms and the L1014F kdr allele in individual mosquitoes., Results: Resistance, at various degrees was detected in both areas of Adzopé. Overall, populations of An. gambiae at both sites surveyed showed equivalent frequency of the L1014F kdr allele (0.67) but for all tested pyrethroids, there were significantly higher survival rates for mosquitoes from Tsassodji (32-58%) than those from Port-Bouët (3-32%) (p < 0.001), indicating the implication of resistance mechanisms other than kdr alone. During the survey period (May-June) in this forested area of Côte d'Ivoire, An. gambiae s. s. found were exclusively of the M form and were apparently selected for pyrethroid resistance through agricultural and household usage of insecticides., Conclusion: Prior to LLINs scaling up in Eastern Côte d'Ivoire, resistance was largely present at various levels in An. gambiae. Underlying mechanisms included the high frequency of the L1014F kdr mutation and other unidentified components, probably metabolic detoxifiers. Their impact on the efficacy of the planned strategy (LLINs) in the area should be investigated alongside careful monitoring of the trend in that resistance over time. The need for alternative insecticides to supplement or replace pyrethroids on nets must be stressed.

Background: At Yaokoffikro field site near Bouaké, in central Côte d'Ivoire, a group of experimental huts built in 1996 served over many years for the evaluation of insecticides against highly resistant mosquitoes. Breeding sites of mosquitoes and selection pressure in the area were maintained by local farming practices until a war broke out in September 2002. Six years after the crisis, we conducted bioassays and biochemical analysis to update the resistance status of Anopheles gambiae s.s. populations and detect other potential mechanisms of resistance that might have evolved., Methods: An. gambiae s.s. larvae from Yaokoffikro were collected in breeding sites and reared to adults. Resistance status of this population to insecticides was assessed using WHO bioassay test kits for adult mosquitoes with seven insecticides: two pyrethroids, a pseudo-pyrethroid, an organochloride, two carbamates and an organophosphate.Molecular and biochemical assays were carried out to identify the L1014F kdr and ace-1R alleles in individual mosquitoes and to detect potential increase in mixed function oxidases (MFO), non-specific esterases (NSE) and glutathione S-transferases (GST) activity., Results: High pyrethroids, DDT and carbamate resistance was confirmed in An. gambiae s.s. populations from Yaokoffikro. Mortality rates were less than 70% with pyrethroids and etofenprox, 12% with DDT, and less than 22% with the carbamates. Tolerance to fenitrothion was observed, with 95% mortality after 24 h.PCR analysis of samples from the site showed high allelic frequency of the L1014F kdr (0.94) and the ace-1R (0.50) as before the crisis. In addition, increased activity of NSE, GST and to a lesser extent MFO was found relative to the reference strain Kisumu. This was the first report detecting enhanced activity of these enzymes in An. gambiae s.s from Yaokoffikro, which could have serious implications in detoxification of insecticides. Their specific roles in resistance should be investigated using additional tools., Conclusion: The insecticide resistance profile at Yaokoffikro appears multifactorial. The site presents a unique opportunity to evaluate its impact on the protective efficacy of insecticidal products as well as new tools to manage these complex mechanisms. It calls for innovative research on the behaviour of the local vector, its biology and genetics that drive resistance.

Background: Neither indoor residual spraying (IRS) nor long-lasting insecticidal nets (LLINs) are able to fully interrupt transmission in holoendemic Africa as single interventions. The combining of IRS and LLINs presents an opportunity for improved control and management of pyrethroid resistance through the simultaneous presentation of unrelated insecticides., Method: Chlorfenapyr IRS and a pyrethroid-impregnated polyester LLIN (WHO approved) were tested separately and together in experimental huts in southern Benin against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus. The bed nets were deliberately holed with either six or 80 holes to examine the effect of increasing wear and tear on protectiveness. Anopheles gambiae were genotyped for the kdr gene to assess the combination's potential to prevent the selection of pyrethroid resistance., Results: The frequency of kdr was 84%. The overall mortality rates of An. gambiae were 37% and 49% with the six-hole and 80-hole LLINs, respectively, and reached 57% with chlorfenapyr IRS. Overall mortality rates were significantly higher with the combination treatments (82-83%) than with the LLIN or IRS individual treatments. Blood feeding (mosquito biting) rates were lowest with the 6-hole LLIN (12%), intermediate with the 80-hole LLIN (32%) and highest with untreated nets (56% with the 6-hole and 54% with the 80-hole nets). Blood feeding (biting) rates and repellency of mosquitoes with the combination of LLIN and chlorfenapyr IRS showed significant improvement compared to the IRS treatment but did not differ from the LLIN treatments indicating that the LLINs were the primary agents of personal protection. The combination killed significantly higher proportions of Cx. quinquefasciatus (51%, 41%) than the LLIN (15%, 13%) or IRS (32%) treatments., Conclusion: The chlorfenapyr IRS component was largely responsible for controlling pyrethroid-resistant mosquitoes and the LLIN component was largely responsible for blood feeding inhibition and personal protection. Together, the combination shows potential to provide additional levels of transmission control and personal protection against pyrethroid-resistant mosquitoes, thereby justifying the additional resources required. Chlorfenapyr has potential to manage pyrethroid resistance in the context of an expanding LLIN/IRS strategy.

Background: Insecticide treatment of nets, curtains or walls and ceilings of houses represent the primary means for malaria prevention worldwide. Direct personal protection of individuals and households arises from deterrent and insecticidal activities which divert or kill mosquitoes before they can feed. However, at high coverage, community-level reductions of mosquito density and survival prevent more transmission exposure than the personal protection acquired by using a net or living in a sprayed house., Methods: A process-explicit simulation of malaria transmission was applied to results of 4 recent Phase II experimental hut trials comparing a new mosaic long-lasting insecticidal net (LLIN) which combines deltamethrin and piperonyl butoxide with another LLIN product by the same manufacturer relying on deltamethrin alone., Results: Direct estimates of mean personal protection against insecticide-resistant vectors in Vietnam, Cameroon, Burkina Faso and Benin revealed no clear advantage for combination LLINs over deltamethrin-only LLINs (P = 0.973) unless both types of nets were extensively washed (Relative mean entomologic inoculation rate (EIR) ± standard error of the mean (SEM) for users of combination nets compared to users of deltamethrin only nets = 0.853 ± 0.056, P = 0.008). However, simulations of impact at high coverage (80% use) predicted consistently better impact for the combination net across all four sites (Relative mean EIR ± SEM in communities with combination nets, compared with those using deltamethrin only nets = 0.613 ± 0.076, P < 0.001), regardless of whether the nets were washed or not (P = 0.467). Nevertheless, the degree of advantage obtained with the combination varied substantially between sites and their associated resistant vector populations., Conclusion: Process-explicit simulations of community-level protection, parameterized using locally-relevant experimental hut studies, should be explicitly considered when choosing vector control products for large-scale epidemiological trials or public health programme procurement, particularly as growing insecticide resistance necessitates the use of multiple active ingredients.

Background: Pyrethroid resistant mosquitoes are becoming increasingly common in parts of Africa. It is important to identify alternative insecticides which, if necessary, could be used to replace or supplement the pyrethroids for use on treated nets. Certain compounds of an earlier generation of insecticides, the organophosphates may have potential as net treatments., Methods: Comparative studies of chlorpyrifos-methyl (CM), an organophosphate with low mammalian toxicity, and lambdacyhalothrin (L), a pyrethroid, were conducted in experimental huts in Côte d'Ivoire, West Africa. Anopheles gambiae and Culex quinquefasciatus mosquitoes from the area are resistant to pyrethroids and organophosphates (kdr and insensitive acetylcholinesterase Ace.1R). Several treatments and application rates on intact or holed nets were evaluated, including single treatments, mixtures, and differential wall/ceiling treatments., Results and Conclusion: All of the treatments were effective in reducing blood feeding from sleepers under the nets and in killing both species of mosquito, despite the presence of the kdr and Ace.1R genes at high frequency. In most cases, the effects of the various treatments did not differ significantly. Five washes of the nets in soap solution did not reduce the impact of the insecticides on A. gambiae mortality, but did lead to an increase in blood feeding. The three combinations performed no differently from the single insecticide treatments, but the low dose mixture performed encouragingly well indicating that such combinations might be used for controlling insecticide resistant mosquitoes. Mortality of mosquitoes that carried both Ace.1R and Ace.1S genes did not differ significantly from mosquitoes that carried only Ace.1S genes on any of the treated nets, indicating that the Ace.1R allele does not confer effective resistance to chlorpyrifos-methyl under the realistic conditions of an experimental hut.