Community evaluation of VECTRON™ T500, a broflanilide insecticide, for indoor residual spraying for malaria vector control in central Benin; a two arm non-inferiority cluster randomised trial.

VECTRON™ T500 is a wettable powder IRS formulation of broflanilide, a newly discovered insecticide. We performed a two-arm non-inferiority community randomised evaluation of VECTRON™ T500, compared to Fludora® Fusion against pyrethroid-resistant Anopheles gambiae s.l. in an area of high coverage with pyrethroid-only nets in the Za-Kpota District of central Benin. One round of IRS was applied in all consenting households in the study area. Sixteen clusters were randomised (1:1) to receive VECTRON™ T500 (100 mg/m2 for broflanilide) or Fludora® Fusion (200 mg/m2 for clothianidin and 25 mg/m2 for deltamethrin). Surveys were performed to assess adverse events and the operational feasibility and acceptability of VECTRON™ T500 among spray operators and household inhabitants. Human landing catches were conducted in 6 households every 1-2 months for up to 18 months post-intervention to assess the impact on vector densities, sporozoite rates and entomological inoculation rates. Bottle bioassays were performed to monitor vector susceptibility to pyrethroids, broflanilide and clothianidin. Monthly wall cone bioassays were conducted for 24 months to assess the residual efficacy of the IRS formulations using susceptible and pyrethroid-resistant An. gambiae s.l. A total of 26,562 female mosquitoes were collected during the study, of which 40% were An. gambiae s.l., the main malaria vector in the study area. The vector population showed high intensity pyrethroid resistance but was susceptible to broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle). Using a non-inferiority margin of 50%, vector density indicated by the human biting rate (bites/person/night) was non-inferior in the VECTRON™ T500 arm compared to the Fludora® Fusion arm both indoors (0.846 bites/p/n in Fludora® Fusion arm vs. 0.741 bites/p/n in VECTRON™ T500 arm, IRR 0.54, 95% CI 0.22-1.35, p = 0.150) and outdoors (0.691 bites/p/n in Fludora® Fusion arm vs. 0.590 bites/p/n in VECTRON™ T500 clusters, IRR 0.75, 95% CI 0.41-1.38, p = 0.297). Sporozoite rates and entomological inoculation rates did not differ significantly between study arms (sporozoite rate: 0.9% vs 1.1%, p = 0. 0.746, EIR: 0.008 vs 0.006 infective bites per person per night, p = 0.589). Cone bioassay mortality with both VECTRON™ T500 and Fludora® Fusion was 100% for 24 months post-IRS application on both cement and mud treated house walls with both susceptible and pyrethroid-resistant strains of An. gambiae s.l. Perceived adverse events reported by spray operators and householders were generally very low (< 6%) in both study arms. VECTRON™ T500 was non-inferior to Fludora® Fusion in reducing the risk of malaria transmission by pyrethroid resistant vectors when applied for IRS in communities in central Benin. The insecticide showed prolonged residual efficacy on house walls, lasting over 24 months and had a high acceptability with homeowners. Community application of VECTRON™ T500 for IRS provides improved and prolonged control of pyrethroid resistant malaria vectors and enhances our capacity to manage insecticide resistance.

Evaluating the attrition, fabric integrity and insecticidal durability of two dual active ingredient nets (Interceptor® G2 and Royal® Guard): methodology for a prospective study embedded in a cluster randomized controlled trial in Benin.

BACKGROUND: Following the World Health Organization (WHO) endorsement of dual active ingredient (AI) nets, an increased uptake of pyrethroid-chlorfenapyr and pyrethroid-pyriproxyfen nets is expected. Studies evaluating their physical and insecticidal durability are essential for making programmatic and procurement decisions. This paper describes the methodology for a prospective study to evaluate the attrition, fabric integrity, insecticidal durability of Interceptor® G2 (alpha-cypermethrin-chlorfenapyr) and Royal Guard® (alpha-cypermethrin-pyriproxyfen), compared to Interceptor® (alpha-cypermethrin), embedded in a 3-arm cluster randomized controlled trial (cRCT) in the Zou Department of Benin. METHODS: Ten clusters randomly selected from each arm of the cRCT will be used for the study. A total of 750 ITNs per type will be followed in 5 study clusters per arm to assess ITN attrition and fabric integrity at 6-, 12-, 24- and 36-months post distribution, using standard WHO procedures. A second cohort of 1800 nets per type will be withdrawn every 6 months from all 10 clusters per arm and assessed for chemical content and biological activity in laboratory bioassays at each time point. Alpha-cypermethrin bioefficacy in Interceptor® and Royal Guard® will be monitored in WHO cone bioassays and tunnel tests using the susceptible Anopheles gambiae Kisumu strain. The bioefficacy of the non-pyrethroid insecticides (chlorfenapyr in Interceptor® G2 and pyriproxyfen in Royal Guard®) will be monitored using the pyrethroid-resistant Anopheles coluzzii Akron strain. Chlorfenapyr activity will be assessed in tunnel tests while pyriproxyfen activity will be assessed in cone bioassays in terms of the reduction in fertility of blood-fed survivors observed by dissecting mosquito ovaries. Nets withdrawn at 12, 24 and 36 months will be tested in experimental hut trials within the cRCT study area against wild free-flying pyrethroid resistant An. gambiae sensu lato to investigate their superiority to Interceptor® and to compare them to ITNs washed 20 times for experimental hut evaluation studies. Mechanistic models will also be used to investigate whether entomological outcomes with each dual ITN type in experimental hut trials can predict their epidemiological performance in the cRCT. CONCLUSION: This study will provide information on the durability of two dual AI nets (Interceptor® G2 and Royal Guard®) in Benin and will help identify suitable methods for monitoring the durability of their insecticidal activity under operational conditions. The modelling component will determine the capacity of experimental hut trials to predict the epidemiological performance of dual AI nets across their lifespan.

Investigating discriminating concentrations for monitoring susceptibility to broflanilide and cross resistance to other insecticide classes in Anopheles gambiae sensu lato, using the new WHO bottle bioassay method.

BACKGROUND: Broflanilide is a new insecticide being developed for malaria vector control. As new insecticide chemistries become available, strategies to preserve the susceptibility of local malaria vectors and extend their useful life need to be considered before large scale deployment. This requires the development of appropriate testing procedures and identification of suitable discriminating concentrations for monitoring susceptibility in wild vector populations to facilitate decision making by control programmes. METHODS: Dose-response WHO bottle bioassays were conducted using the insecticide-susceptible Anopheles gambiae s.s. Kisumu strain to determine a discriminating concentration of broflanilide. Bioassays were performed without the adjuvant Mero® and with two concentrations of Mero® (500 ppm and 800 ppm) to investigate its impact on the discriminating concentration of the insecticide. Probit analysis was used to determine the lethal doses at 50% (LC50) and 99% (LC99) at 24-, 48- and 72-hours post-exposure. Cross-resistance to broflanilide and pyrethroids, DDT, dieldrin and carbamates, was investigated using An. gambiae s.l. Covè and An. coluzzii Akron strains. The susceptibility of wild pyrethroid-resistant mosquitoes from communities in Southern Benin to broflanilide was assessed using the estimated discriminating concentrations. RESULTS: Broflanilide induced a dose-dependent and delayed mortality effect. Mortality rates in bottles treated without Mero® were <80% using the range of broflanilide doses tested (0-100 µg/bottle) leading to high and unreliable estimates of LC99 values. The discriminating concentrations defined as 2XLC99 at 72h post exposure were estimated to be 2.2 µg/bottle with 800 ppm of Mero® and 6.0 µg/bottle with 500 ppm of Mero®. Very low resistance ratios (0.6-1.2) were determined with the insecticide resistant An. gambiae s.l. Covè and An. coluzzii Akron strains suggesting the absence of cross-resistance via the mechanisms of resistance to pyrethroids, DDT, dieldrin and carbamates they possess. Bottle bioassays performed with broflanilide at both discriminating concentrations of 6 µg/bottle with 500 ppm of Mero® and 2.2 µg/bottle with 800 ppm of Mero®, showed susceptibility of wild highly pyrethroid-resistant An. gambiae s.l. from villages in Southern Benin. CONCLUSION: We determined discriminating concentrations for monitoring susceptibility to broflanilide in bottle bioassays, using susceptible An. gambiae vectors. Using the estimated discriminating concentrations, we showed that wild pyrethroid-resistant populations of An. gambiae s.l. from southern Benin were fully susceptible to the insecticide. Broflanilide also shows potential to be highly effective against An. gambiae s.l. vector populations that have developed resistance to other public health insecticides.

VECTRON™ T500, a new broflanilide insecticide for indoor residual spraying, provides prolonged control of pyrethroid-resistant malaria vectors.

BACKGROUND: Broflanilide is a newly discovered insecticide with a novel mode of action targeting insect γ-aminobutyric acid receptors. The efficacy of VECTRON™ T500, a wettable powder formulation of broflanilide, was assessed for IRS against wild pyrethroid-resistant malaria vectors in experimental huts in Benin. METHODS: VECTRON™ T500 was evaluated at 100 mg/m2 in mud and cement-walled experimental huts against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) in Covè, southern Benin, over 18 months. A direct comparison was made with Actellic® 300CS, a WHO-recommended micro-encapsulated formulation of pirimiphos-methyl, applied at 1000 mg/m2. The vector population at Covè was investigated for susceptibility to broflanilide and other classes of insecticides used for vector control. Monthly wall cone bioassays were performed to assess the residual efficacy of VECTRON™ T500 using insecticide susceptible An. gambiae Kisumu and pyrethroid-resistant An. gambiae s.l. Covè strains. The study complied with OECD principles of good laboratory practice. RESULTS: The vector population at Covè was resistant to pyrethroids and organochlorines but susceptible to broflanilide and pirimiphos-methyl. A total of 23,171 free-flying wild pyrethroid-resistant female An. gambiae s.l. were collected in the experimental huts over 12 months. VECTRON™ T500 induced 56%-60% mortality in wild vector mosquitoes in both cement and mud-walled huts. Mortality with VECTRON™ T500 was 62%-73% in the first three months and remained > 50% for 9 months on both substrate-types. By comparison, mortality with Actellic® 300CS was very high in the first three months (72%-95%) but declined sharply to < 40% after 4 months. Using a non-inferiority margin defined by the World Health Organization, overall mortality achieved with VECTRON™ T500 was non-inferior to that observed in huts treated with Actellic® 300CS with both cement and mud wall substrates. Monthly in situ wall cone bioassay mortality with VECTRON™ T500 also remained over 80% for 18 months but dropped below 80% with Actellic® 300CS at 6-7 months post spraying. CONCLUSION: VECTRON™ T500 shows potential to provide substantial and prolonged control of malaria transmitted by pyrethroid-resistant mosquito vectors when applied for IRS. Its addition to the current list of WHO-approved IRS insecticides will provide a suitable option to facilitate rotation of IRS products with different modes of action.

Pirikool® 300 CS, a new long-lasting capsule suspension formulation of the organophosphate insecticide pirimiphos-methyl for indoor residual spraying against pyrethroid-resistant malaria vectors.

BACKGROUND: Indoor residual spraying (IRS) using a capsule suspension formulation of the organophosphate insecticide, pirimiphos-methyl, has provided substantial malaria control in many communities in Africa. However, only one brand of this product has been recommended by the World Health Organisation for IRS. To help increase the diversity of the portfolio of IRS insecticides and offer suitable options to procurers and malaria vector control programmes, additional product brands of this highly effective and long-lasting insecticide formulation for IRS will be needed. METHODS: We evaluated the efficacy of Pirikool® 300CS, a new capsule suspension formulation of pirimiphos-methyl developed by Tianjin Yorkool, International Trading, Co., Ltd in standard WHO laboratory bioassays and experimental hut studies. The efficacy of the insecticide applied at 1000mg/m2 was assessed in laboratory bioassays for 6 months on cement, plywood and mud block substrates and for 12 months in cement and mud-walled experimental huts against wild free-flying pyrethroid-resistant Anopheles gambiae sensu lato in Covè, Benin. Actellic® 300CS, a WHO-recommended capsule suspension formulation of pirimiphos-methyl was also tested. WHO cylinder tests were performed to determine the frequency of insecticide resistance in the wild vector population during the hut trial. RESULTS: The vector population at the hut station was resistant to pyrethroids but susceptible to pirimiphos-methyl. Overall mortality rates of wild free-flying pyrethroid-resistant An. gambiae (s.l.) entering Pirikool®300CS treated experimental huts during the 12-month trial were 86.7% in cement-walled huts and 88% in mud-walled huts. Mortality of susceptible An. gambiae (Kisumu) and pyrethroid-resistant An. gambiae s.l. (Covè) mosquitoes in monthly wall cone bioassays on Pirikool® 300CS treated hut walls remained over 80% for 10-12 months. The laboratory bioassays corroborated the hut findings with Pirikool® 300CS on mud and wood block substrates but not on cement block substrates. CONCLUSION: Indoor residual spraying with Pirikool® 300CS induced high and prolonged mortality of wild pyrethroid-resistant malaria vectors for 10-12 months. Addition of Pirikool®300CS to the current portfolio of IRS insecticides will provide an extra choice of microencapsulated pirimiphos-methyl for IRS.

Pyrethroid-piperonyl butoxide (PBO) nets reduce the efficacy of indoor residual spraying with pirimiphos-methyl against pyrethroid-resistant malaria vectors.

Pirimiphos-methyl is a pro-insecticide requiring activation by mosquito cytochrome P450 enzymes to induce toxicity while PBO blocks activation of these enzymes in pyrethroid-resistant vector mosquitoes. PBO may thus antagonise the toxicity of pirimiphos-methyl IRS when combined with pyrethroid-PBO ITNs. The impact of combining Olyset Plus and PermaNet 3.0 with Actellic 300CS IRS was evaluated against pyrethroid-resistant Anopheles gambiae s.l. in two parallel experimental hut trials in southern Benin. The vector population was resistant to pyrethroids and PBO pre-exposure partially restored deltamethrin toxicity but not permethrin. Mosquito mortality in experimental huts was significantly improved in the combinations of bendiocarb IRS with pyrethroid-PBO ITNs (33-38%) compared to bendiocarb IRS alone (14-16%, p < 0.001), demonstrating an additive effect. Conversely, mortality was significantly reduced in the combinations of pirimiphos-methyl IRS with pyrethroid-PBO ITNs (55-59%) compared to pirimiphos-methyl IRS alone (77-78%, p < 0.001), demonstrating evidence of an antagonistic effect when both interventions are applied in the same household. Mosquito mortality in the combination was significantly higher compared to the pyrethroid-PBO ITNs alone (55-59% vs. 22-26% p < 0.001) showing potential of pirimiphos-methyl IRS to enhance vector control when deployed to complement pyrethroid-PBO ITNs in an area where PBO fails to fully restore susceptibility to pyrethroids.

Efficacy of broflanilide (VECTRON T500), a new meta-diamide insecticide, for indoor residual spraying against pyrethroid-resistant malaria vectors.

The rotational use of insecticides with different modes of action for indoor residual spraying (IRS) is recommended for improving malaria vector control and managing insecticide resistance. Insecticides with new chemistries are urgently needed. Broflanilide is a newly discovered insecticide under consideration. We investigated the efficacy of a wettable powder (WP) formulation of broflanilide (VECTRON T500) for IRS on mud and cement wall substrates in laboratory and experimental hut studies against pyrethroid-resistant malaria vectors in Benin, in comparison with pirimiphos-methyl CS (Actellic 300CS). There was no evidence of cross-resistance to pyrethroids and broflanilide in CDC bottle bioassays. In laboratory cone bioassays, broflanilide WP-treated substrates killed > 80% of susceptible and pyrethroid-resistant An. gambiae sl for 6-14 months. At application rates of 100 mg/m2 and 150 mg/m2, mortality of wild pyrethroid-resistant An. gambiae sl entering experimental huts in Covè, Benin treated with VECTRON T500 was similar to pirimiphos-methyl CS (57-66% vs. 56%, P > 0.05). Throughout the 6-month hut trial, monthly wall cone bioassay mortality on VECTRON T500 treated hut walls remained > 80%. IRS with broflanilide shows potential to significantly improve the control of malaria transmitted by pyrethroid-resistant mosquito vectors and could thus be a crucial addition to the current portfolio of IRS insecticides.

Which indoor residual spraying insecticide best complements standard pyrethroid long-lasting insecticidal nets for improved control of pyrethroid resistant malaria vectors?

BACKGROUND: Where resources are available, non-pyrethroid IRS can be deployed to complement standard pyrethroid LLINs with the aim of achieving improved vector control and managing insecticide resistance. The impact of the combination may however depend on the type of IRS insecticide deployed. Studies comparing combinations of pyrethroid LLINs with different types of non-pyrethroid IRS products will be necessary for decision making. METHODS: The efficacy of combining a standard pyrethroid LLIN (DuraNet®) with IRS insecticides from three chemical classes (bendiocarb, chlorfenapyr and pirimiphos-methyl CS) was evaluated in an experimental hut trial against wild pyrethroid-resistant Anopheles gambiae s.l. in Cové, Benin. The combinations were also compared to each intervention alone. WHO cylinder and CDC bottle bioassays were performed to assess susceptibility of the local An. gambiae s.l. vector population at the Cové hut site to insecticides used in the combinations. RESULTS: Susceptibility bioassays revealed that the vector population at Cové, was resistant to pyrethroids (<20% mortality) but susceptible to carbamates, chlorfenapyr and organophosphates (≥98% mortality). Mortality of wild free-flying pyrethroid resistant An. gambiae s.l. entering the hut with the untreated net control (4%) did not differ significantly from DuraNet® alone (8%, p = 0.169). Pirimiphos-methyl CS IRS induced the highest mortality both on its own (85%) and in combination with DuraNet® (81%). Mortality with the DuraNet® + chlorfenapyr IRS combination was significantly higher than each intervention alone (46% vs. 33% and 8%, p<0.05) demonstrating an additive effect. The DuraNet® + bendiocarb IRS combination induced significantly lower mortality compared to the other combinations (32%, p<0.05). Blood-feeding inhibition was very low with the IRS treatments alone (3-5%) but increased significantly when they were combined with DuraNet® (61% - 71%, p<0.05). Blood-feeding rates in the combinations were similar to the net alone. Adding bendiocarb IRS to DuraNet® induced significantly lower levels of mosquito feeding compared to adding chlorfenapyr IRS (28% vs. 37%, p = 0.015). CONCLUSIONS: Adding non-pyrethroid IRS to standard pyrethroid-only LLINs against a pyrethroid-resistant vector population which is susceptible to the IRS insecticide, can provide higher levels of vector mosquito control compared to the pyrethroid net alone or IRS alone. Adding pirimiphos-methyl CS IRS may provide substantial improvements in vector control while adding chlorfenapyr IRS can demonstrate an additive effect relative to both interventions alone. Adding bendiocarb IRS may show limited enhancements in vector control owing to its short residual effect.

Efficacy of Fludora® Fusion (a mixture of deltamethrin and clothianidin) for indoor residual spraying against pyrethroid-resistant malaria vectors: laboratory and experimental hut evaluation.

BACKGROUND: A new generation of IRS insecticides which can provide improved and prolonged control of pyrethroid-resistant malaria vector populations are being developed. Fludora® Fusion is a new IRS insecticide containing a mixture of deltamethrin and clothianidin, a neonicotinoid. METHODS: The efficacy of Fludora® Fusion IRS was evaluated over 11-12 months on concrete and mud substrates in laboratory bioassays and experimental huts against wild free-flying pyrethroid-resistant Anopheles gambiae (sensu lato) in Cové, Benin. A comparison was made with the two active ingredients of the mixture; clothianidin and deltamethrin, applied alone. CDC bottle bioassays were also performed to investigate resistance to clothianidin in the wild vector population. RESULTS: Fludora® Fusion induced > 80% laboratory cone bioassay mortality with both susceptible and pyrethroid-resistant An. gambiae (s.l.) for 7-9 months on concrete block substrates and 12 months on mud block substrates. The vector population at the experimental hut site was fully susceptible to clothianidin in CDC bottle bioassays. Overall mortality rates of wild free-flying pyrethroid-resistant An. gambiae (s.l.) entering the experimental huts during the 11-month trial were < 15% with deltamethrin and significantly higher with Fludora® Fusion (69-71%) and clothianidin alone (72-78%). Initial high experimental hut mortality rates with Fludora® Fusion (> 80%) only declined by 50% after 8 months. Monthly in situ wall cone bioassay mortality of susceptible mosquitoes was > 80% for 9-12 months with Fludora® Fusion and clothianidin alone. Fludora® Fusion induced significantly higher levels of early exiting of mosquitoes compared to clothianidin alone (55-60% vs 37-38%, P < 0.05). CONCLUSIONS: Indoor residual spraying with Fludora® Fusion induced high and prolonged mortality of wild pyrethroid-resistant malaria vectors for 7-10 months mostly due to the clothianidin component and substantial early exiting of mosquitoes from treated huts due to the pyrethroid component. Fludora® Fusion is an important addition to the current portfolio of IRS insecticides with the potential to significantly reduce transmission of malaria by pyrethroid-resistant mosquito vectors.

Efficacy of Royal Guard, a new alpha-cypermethrin and pyriproxyfen treated mosquito net, against pyrethroid-resistant malaria vectors.

Royal Guard is a new insecticide-treated bed-net incorporated with a mixture of alpha-cypermethrin and pyriproxyfen (an insect growth regulator). We assessed its efficacy and wash-resistance in laboratory and experimental hut studies following WHO guidelines. Mosquitoes that survived exposure to the net were kept in separate oviposition chambers and observed for the reproductive effects of pyriproxyfen. In laboratory assays, Royal Guard induced > 80% mortality and > 90% blood-feeding inhibition of An. gambiae sl mosquitoes before and after 20 standardised washes and sterilised blood-fed mosquitoes which remained alive after exposure to the net. In an experimental hut trial against wild free-flying pyrethroid-resistant An. gambiae sl in Cové Benin, Royal Guard through the pyrethroid component induced comparable levels of mortality and blood-feeding inhibition to a standard pyrethroid-only treated net before and after 20 washes and sterilised large proportions of surviving blood-fed female mosquitoes through the pyriproxyfen component; Royal Guard induced 83% reduction in oviposition and 95% reduction in offspring before washing and 25% reduction in oviposition and 50% reduction in offspring after 20 washes. Royal Guard has the potential to improve malaria vector control and provide better community protection against clinical malaria in pyrethroid-resistant areas compared to standard pyrethroid-only LLINs.

Indoor spraying with chlorfenapyr (a pyrrole insecticide) provides residual control of pyrethroid-resistant malaria vectors in southern Benin.

BACKGROUND: New classes of insecticides with novel modes of action, which can provide effective and prolonged control of insecticide-resistant malaria vector populations, are urgently needed for indoor residual spraying. Such insecticides can be included in a rotation plan to manage and prevent further development of resistance in mosquito vectors of malaria. Chlorfenapyr, a novel pyrrole insecticide with a unique mode of action, is being developed as a long-lasting IRS formulation. METHODS: The efficacy of several formulations of chlorfenapyr alone and as mixtures with alpha-cypermethrin were evaluated in an experimental hut trial against wild pyrethroid-resistant Anopheles gambiae sensu lato in Cové, Benin, in an attempt to identify the most effective and long-lasting formulations for IRS. The trial lasted 12 months. A comparison was made with alpha-cypermethrin and bendiocarb formulations. CDC bottle bioassays were performed to investigate cross-resistance to chlorfenapyr in the local vector population. RESULTS: Mortality rates in World Health Organization (WHO) cylinder bioassays were < 5% with pyrethroids due to high levels of pyrethroid resistance, but > 95% with bendiocarb thus confirming susceptibility to carbamates in the vector population. CDC bottle bioassays showed no cross-resistance between pyrethroids and chlorfenapyr. Overall mortality of free-flying mosquitoes entering the experimental huts over the 12-month trial was 4% with alpha-cypermethrin and 12% with bendiocarb. The chlorfenapyr solo-formulations induced significantly higher levels of mortality (38-46%) compared to the bendiocarb (12% P < 0.001) and to the mixture formulations (18-22%, P < 0.05). The original Sylando 240SC formulation of chlorfenapyr was more efficacious than all other novel chlorfenapyr formulations tested. Bendiocarb induced > 80% mortality in the first month, but this declined sharply to < 20% by the third month while the mortality rates achieved with the chlorfenapyr formulations (38-46%) were persistent lasting 7-10 months. The mixtures induced significantly lower percentage mortality than chlorfenapyr-solo formulations. Wall cone bioassays only showed mortality rates that were consistent with chlorfenapyr IRS treated huts when the exposure time was increased to 2 h. CONCLUSION: Indoor residual spraying with chlorfenapyr (Sylando® 240SC) provides moderate but prolonged control of pyrethroid-resistant malaria vectors compared to pyrethroid and bendiocarb IRS. Wall cone bioassays on chlorfenapyr-treated walls required longer exposure times of 2 h than the customary 30 min indicating that WHO guidelines on residual cone bioassays need to be more insecticide-specific.

Resistance status of Anopheles gambiae s.l. to insecticides following the 2011 mass distribution campaign of long-lasting insecticidal nets (LLINs) in the Plateau Department, south-eastern Benin.

BACKGROUND: In 2011, Benin's National Malaria Control Programme (NMCP) organized a nationwide mass distribution campaign of LLINs throughout the country. Following this intervention, it was important to assess whether the level of susceptibility of malaria vectors to insecticides had remained the same as compared to the pre-intervention period. The current study investigated this. METHODS: Larval collections were conducted in Ifangni, Sakété, Pobè and Kétou districts located in Plateau department, Southeastern Benin before (2009) and after (2012-2013) LLIN distribution. Anopheles gambiae sensu lato (s.l.) larvae from the 4 study districts were reared to adulthood and WHO susceptibility tests were conducted. The insecticides tested were deltamethrin (0.05%), permethrin (0.75%), bendiocarb (0.1%) and DDT (4%). Molecular species identification as well as, the characterization of the kdr L1014F mutation were also performed in the An. gambiae s.l. complex using PCR method. RESULTS: Overall, a significant decrease in mortality rates of An. gambiae s.l. to deltamethrin (0.05%), permethrin (0.75%) and DDT (4%) was observed post-LLIN distribution, respectively: (100% vs 80.9%, p < 0.0001), (77.5% vs 70%, p = 0.01) and, (47.8% vs 4.4%, p < 0.0001). By contrast, susceptibility of vectors to bendiocarb (0.1%) remained the same (100% mortality in the WHO susceptibility tube tests) pre- and post-intervention. An increase in the kdr L1014F frequency was observed post-LLIN distribution [F(kdr) = 0.91)] compared to the pre-intervention period [F(kdr) = 0.56], p < 0.0001. Anopheles coluzzii and An. gambiae were the two molecular species identified in the study area. CONCLUSION: The decrease susceptibility to pyrethroids and DDT as well as, the increase in the frequency of the kdr L1014F mutation after the intervention stressed at the time, the need for the development and implementation of effective insecticide resistance management strategies. At present, an update of the vectors resistance status in the area is also necessary for decision-making.

Indoor residual spraying with a mixture of clothianidin (a neonicotinoid insecticide) and deltamethrin provides improved control and long residual activity against pyrethroid resistant Anopheles gambiae sl in Southern Benin.

INTRODUCTION: There is an urgent need for new insecticides for indoor residual spraying (IRS) which can provide improved and prolonged control of malaria vectors that have developed resistance to existing insecticides. The neonicotinoid, clothianidin represents a class of chemistry new to public health. Clothianidin acts as an agonist on nicotinic acetyl choline receptors. IRS with a mixture of Clothianidin and another WHO approved insecticide such as deltamethrin could provide improved control of insecticide resistant malaria vector populations and serve as a tool for insecticide resistance management. METHODS: The efficacy and residual activity of a novel IRS mixture of deltamethrin and clothianidin was evaluated against wild pyrethroid resistant An. gambiae sl in experimental huts in Cove, Benin. Two application rates of the mixture were tested and comparison was made with clothianidin and deltamethrin applied alone. To assess the residual efficacy of the treatments on different local wall substrates, the inner walls of the experimental huts were covered with either cement, mud or plywood. RESULTS: Clothianidin demonstrated a clear delayed expression in mortality of wild pyrethroid resistant An. gambiae sl in the experimental huts which reached its full effect 120 hours after exposure. Overall mortality over the 12-month hut trial was 15% in the control hut and 24-29% in the deltamethrin-treated huts. The mixture of clothianidin 200mg/m2 and deltamethrin 25mg/m2 induced high overall hut mortality rates (87% on mud walls, 82% on cement walls and 61% on wooden walls) largely due to the clothianidin component and high hut exiting rates (67-76%) mostly due to the deltamethrin component. Mortality rates remained >80% for 8-9 months on mud and cement walls. The residual activity trend was confirmed by results from monthly in situ cone bioassays with laboratory susceptible An. gambiae Kisumu strain. CONCLUSION: IRS campaigns with the mixture of clothianidin plus deltamethrin have the potential to provide prolonged control of malaria transmitted by pyrethroid resistant mosquito populations.

Relative performance of indoor vector control interventions in the Ifakara and the West African experimental huts.

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.

Insecticide resistance profile of Anopheles gambiae from a phase II field station in Cové, southern Benin: implications for the evaluation of novel vector control products.

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.