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.

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.

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.

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.

Efficacy of the Olyset Duo net against insecticide-resistant mosquito vectors of malaria.

Olyset Duo is a new long-lasting insecticidal net treated with permethrin (a pyrethroid) and pyriproxyfen, an insect growth regulator that disrupts the maturation of oocytes in mosquitoes exposed to the net. We tested the Olyset Duo net against pyrethroid-resistant Anopheles gambiae mosquitoes, which transmit malaria parasites, in laboratory bioassays and in a trial in Benin using experimental huts that closely resemble local habitations. Host-seeking mosquitoes that entered to feed were free to contact the occupied nets and were collected the next morning from exit traps. Surviving blood-fed mosquitoes were observed for effects on reproduction. Control nets were treated with pyrethroid only or pyriproxyfen only, and nets were tested unwashed and after 20 standardized washes. The Olyset Duo net showed improved efficacy and wash resistance relative to the pyrethroid-treated net in terms of mosquito mortality and prevention of blood feeding. The production of offspring among surviving blood-fed A. gambiae in the hut trial was reduced by the pyriproxyfen-treated net and the Olyset Duo net both before washing (90 and 71% reduction, respectively) and after washing (38 and 43% reduction, respectively). The degree of reproductive suppression in the hut trial was predicted by laboratory tunnel tests but not by cone bioassays. The overall reduction in reproductive rate of A. gambiae with the Olyset Duo net in the trial was 94% with no washing and 78% after 20 washes. The Olyset Duo net has the potential to provide community control of mosquito populations and reduce malaria transmission in areas of high insecticide resistance.