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

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  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

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  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

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  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  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

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

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

Mosquito Shield™, a transfluthrin passive emanator, protects against pyrethroid-resistant Anopheles gambiae sensu lato in central Benin.

BACKGROUND: Spatial repellents can provide personal and household protection against biting vector mosquitoes by volatizing repellents into the air within a given area. Mosquito Shield™ is a transfluthrin passive emanator undergoing evaluation for malaria control. Studies evaluating its entomological impact against different local malaria vector populations would help guide its deployment in endemic countries. METHODS: A two-arm single-blinded small-scale household randomised entomological trial was conducted to assess the impact of Mosquito Shield™ on the human landing rate of wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) vector mosquitoes in houses in the Ganhoua village of the Zakpota District of central Benin. From a total of 30 houses, 15 were randomly allocated to receive Mosquito Shield™, while the remainder received a placebo product. The trial lasted through the life of the Mosquito Shield™ product (32 days). Mosquito sampling was performed by human landing catches at baseline and at 6 timepoints post-intervention (days 0-1, 7-8, 14-15, 21-22, 28-29 and 31-32). Collections were performed for 2 nights at each sampling time point. WHO cylinder bioassays were conducted during the trial with F1 An. gambiae s.l. mosquitoes that emerged from larvae from the study area to assess the intensity of resistance to pyrethroids in the wild vector population. RESULTS: The vector population in the study area showed a high intensity of resistance to pyrethroids. Baseline An. gambiae s.l. human landing rates were similar in houses in both study arms before product application (11.53/person/night vs 11.67/person/night, p > 0.05). A total of 5736 mosquitoes were collected in the placebo control arm and 3862 in the Mosquito Shield™ arm post-intervention. Overall An. gambiae s.l. post-intervention human landing rates were significantly lower in houses in the Mosquito Shield™ arm (18.13/person/night) compared to the houses in the placebo control arm (26.84/person/night, IRR = 0.658, p < 0.001). Over the lifespan of the product, Mosquito Shield™ provided a significant protective efficacy of 34.2% (22.1-44.4%, p < 0.001) against wild pyrethroid-resistant An. gambiae s.l. vectors compared to the placebo. Human landing rates of other nuisance vector mosquito species (Culex and Mansonia) were also reduced in houses treated with Mosquito Shield™ compared to the placebo. CONCLUSION: Mosquito Shield™, a transfluthrin passive emanator, provided significant protection against pyrethroid-resistant malaria vectors to households in Benin. The spatial repellent shows potential to reduce malaria transmission by pyrethroid-resistant An. gambiae s.l. vector mosquitoes and cover gaps in malaria control when deployed to complement existing vector control interventions

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

Malaria prevalence and transmission in the Zakpota sub-district of central Benin: baseline characteristics for a community randomised trial of a new insecticide for indoor residual spraying.

BACKGROUND: Malaria transmission is known to be perennial and heterogeneous in Benin. Studies assessing local malaria prevalence, transmission levels and vector characteristics are critical for designing, monitoring and evaluating new vector control interventions in community trials. We conducted a study in the Zakpota sub-district of central Benin to collect baseline data on household characteristics, malaria prevalence, vector characteristics and transmission dynamics in preparation for a randomised controlled trial to evaluate the community impact of VECTRON™ T500, a new broflanilide indoor residual spraying (IRS) product. METHODS: A total of 480 children under 5 years of age from the 15 villages of the sub-district were tested for malaria by rapid diagnostic tests (RDTs). Mosquitoes were collected by human landing catches (HLCs), pyrethrum spray catches (PSCs) and Centers for Disease Control and Prevention miniature light traps (CDC-LTs) in selected houses in each village to assess vector density, composition, vector infectivity and prevalence of insecticide resistance markers. Bioassays were performed to detect vector susceptibility to pyrethroids, broflanilide (6 µg/bottle) and clothianidin (90 µg/bottle). RESULTS: A total of 9080 households were enumerated in the 15 study villages. Insecticide-treated net (ITN) usage was > 90%, with 1-2 ITNs owned per household. Houses were constructed mainly with cement (44%) and mud (38%) substrates or a mixture of cement and mud (18%), and 60% of them had open eaves. The overall prevalence of P. falciparum infection was 19% among surveyed children: 20% among females and 18% among males. The haemoglobin rate showed an anaemia (< 11 g/dl) prevalence of 66%. Anopheles coluzzii and An. gambiae sensu stricto (s.s.) were the two vector species present at an overall proportion of 46% versus 54%, respectively. The human biting rate was 2.3 bites per person per night (b/p/n) and biting occurred mostly indoors compared with outdoors (IRR = 0.776; P = 0.001). The overall proportion of outdoor biting was 44% and exceeded indoor biting in three villages. The sporozoite rate was 2% with a combined yearly entomological inoculation rate (EIR) of 16.1 infected bites per person per year (ib/p/y). There was great variability in malaria transmission risk across the villages, with EIR ranging from 0 to 29.3 ib/p/y. The vector population showed a high intensity of resistance to pyrethroids across the study villages but was largely susceptible to broflanilide and clothianidin. CONCLUSIONS: This study found high levels of malaria prevalence, vector density and transmission in the Zakpota sub-district despite the wide use of insecticide-treated nets. The vector population was mostly indoor resting and showed a high intensity of pyrethroid resistance but was generally fully susceptible to broflanilide. These findings demonstrated the suitability of the study area for the assessment of VECTRON™ T500 in a community randomised trial