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

Combining organophosphate-treated wall linings and long-lasting insecticidal nets fails to provide additional control over long-lasting insecticidal nets alone against multiple insecticide-resistant <i>Anopheles gambiae</i> in Côte d’Ivoire: an experimental hut trial

&lt;b&gt;Background&lt;/b&gt; Insecticide-treated wall lining (ITWL) is a new concept in malaria vector control. Some &lt;i&gt;Anopheles gambiae&lt;/i&gt; 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.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Methods&lt;/b&gt; 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 &lt;i&gt;An. Gambiae&lt;/i&gt; in Tiassalé, Côte d’Ivoire. Comparison was made to commercial deltamethrin WL and genotypes for &lt;i&gt;kdr&lt;/i&gt; and &lt;i&gt;ace-1R&lt;/i&gt; resistance were monitored.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Results&lt;/b&gt; The &lt;i&gt;kdr&lt;/i&gt; and &lt;i&gt;ace-1R&lt;/i&gt; allele frequencies were 0.83 and 0.44, respectively. &lt;i&gt;Anopheles gambiae&lt;/i&gt; surviving discriminating concentrations of deltamethrin and p-methyl in WHO resistance tests were 57 and 96%, respectively. Mortality of free-flying &lt;i&gt;An. Gambiae&lt;/i&gt; in huts with p-methyl WL and NWH (66 and 50%, respectively) was higher than with pyrethroid WL (32%; P &lt; 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 &lt;i&gt;ace-1R&lt;/i&gt; were more likely to survive exposure to p-methyl WL and NWH. Selection of heterozygote and homozygote &lt;i&gt;ace-1R&lt;/i&gt; or &lt;i&gt;kdr&lt;/i&gt; 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.&lt;p&gt;&lt;/p&gt; &lt;b&gt;Conclusions&lt;/b&gt; Combining p-methyl WL or NWH with LLINs provided no improvement in &lt;i&gt;An. Gambiae&lt;/i&gt; control or personal protection over LLIN alone in southern Côte d’Ivoire; neither did the combination manage resistance. Additional resistance mechanisms to &lt;i&gt;kdr&lt;/i&gt; and &lt;i&gt;ace-1R&lt;/i&gt; 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.&lt;p&gt;&lt;/p&gt

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

Olyset Duo® (a pyriproxyfen and permethrin mixture net): an experimental hut trial against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus in Southern Benin.

BACKGROUND: Alternative compounds which can complement pyrethroids on long-lasting insecticidal nets (LN) in the control of pyrethroid resistant malaria vectors are urgently needed. Pyriproxyfen (PPF), an insect growth regulator, reduces the fecundity and fertility of adult female mosquitoes. LNs containing a mixture of pyriproxyfen and pyrethroid could provide personal protection through the pyrethroid component and reduce vector abundance in the next generation through the sterilizing effect of pyriproxyfen. METHOD: The efficacy of Olyset Duo, a newly developed mixture LN containing pyriproxyfen and permethrin, was evaluated in experimental huts in southern Benin against pyrethroid resistant Anopheles gambiae and Culex quinquefasciatus. Comparison was made with Olyset Net® (permethrin alone) and a LN with pyriproxyfen alone (PPF LN). Laboratory tunnel tests were performed to substantiate the findings in the experimental huts. RESULTS: Overall mortality of wild pyrethroid resistant An. gambiae s.s. was significantly higher with Olyset Duo than with Olyset Net (50% vs. 27%, P = 0.01). Olyset DUO was more protective than Olyset Net (71% vs. 3%, P<0.001). The oviposition rate of surviving blood-fed An. gambiae from the control hut was 37% whereas none of those from Olyset Duo and PPF LN huts laid eggs. The tunnel test results were consistent with the experimental hut results. Olyset Duo was more protective than Olyset Net in the huts against wild pyrethroid resistant Cx. quinquefasciatus although mortality rates of this species did not differ significantly between Olyset Net and Olyset Duo. There was no sterilizing effect on surviving blood-fed Cx. quinquefasciatus with the PPF-treated nets. CONCLUSION: Olyset Duo was superior to Olyset Net in terms of personal protection and killing of pyrethroid resistant An. gambiae, and sterilized surviving blood-fed mosquitoes. Mixing pyrethroid and pyriproxyfen on a LN shows potential for malaria control and management of pyrethroid resistant vectors by preventing further selection of pyrethroid resistant phenotypes

Which intervention is better for malaria vector control: insecticide mixture long-lasting insecticidal nets or standard pyrethroid nets combined with indoor residual spraying?

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

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

The Activityof the Pyrrole Insecticide Chlorfenapyr in Mosquito Bioassay: Towards a More Rational Testing and Screening of Non-neurotoxic Insecticides for Malaria\ud vector control

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. The efficacy of WHO recommended cone, cylinder and tunnel tests was compared for pyrethroids and chlorfenapyr. To establish bioassay exposure times predictive of insecticidetreated 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. 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. Testing according to current WHO guidelines is not suitable for certain types of nonneurotoxic 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 nightactive anophelines is the most reliable bioassay for identifying the toxicity of nove

The Automatic Classification of Pyriproxyfen-Affected Mosquito Ovaries.

Pyriproxyfen (PPF) may become an alternative insecticide for areas where pyrethroid-resistant vectors are prevalent. The efficacy of PPF can be assessed through the dissection and assessment of vector ovaries. However, this reliance on expertise is subject to limitations. We show here that these limitations can be overcome using a convolutional neural network (CNN) to automate the classification of egg development and thus fertility status. Using TensorFlow, a resnet-50 CNN was pretrained with the ImageNet dataset. This CNN architecture was then retrained using a novel dataset of 524 dissected ovary images from An. gambiae s.l. An. gambiae Akron, and An. funestus s.l., whose fertility status and PPF exposure were known. Data augmentation increased the training set to 6973 images. A test set of 157 images was used to measure accuracy. This CNN model achieved an accuracy score of 94%, and application took a mean time of 38.5 s. Such a CNN can achieve an acceptable level of precision in a quick, robust format and can be distributed in a practical, accessible, and free manner. Furthermore, this approach is useful for measuring the efficacy and durability of PPF treated bednets, and it is applicable to any PPF-treated tool or similarly acting insecticide