Community evaluation of the physical and insecticidal durability of DuraNet® Plus, an alpha-cypermethrin and piperonyl butoxide incorporated mosquito net: protocol for a multi-country study in West, Central and East Africa.

BACKGROUND: Pyrethroid-PBO nets have demonstrated improved impact against clinical malaria transmitted by pyrethroid resistant mosquito vectors and are being scaled up across Africa. However very little is known about their physical and insecticidal durability under operational conditions. This study will investigate the attrition, fabric integrity, insecticide content and bioefficacy of DuraNet® Plus, a new WHO prequalified alphacypermethrin and PBO incorporated net developed by Shobikaa Impex Private Limited over 3 years of field use in communities in Benin, Cameroon and Tanzania. METHODS: The study will be conducted in parallel in selected villages in Zakpota District in Benin, Mbalmayo, District in Cameroon and Muheza District in Tanzania. In each country, ~ 1800 households will be recruited and randomised to receive DuraNet® Plus or DuraNet® (a WHO prequalified alphacypermethrin-only ITN). Follow up surveys will be performed at 1 month post distribution to investigate adverse events and subsequently every 6-12 months to assess ITN attrition and fabric integrity following standard WHO procedures. A second cohort of nets will be withdrawn every 6-12 months and assessed for alpha-cypermethrin and PBO content and for entomological activity in laboratory bioassays (cone bioassays and tunnel tests). Alpha-cypermethrin bioefficacy will be monitored using the susceptible Anopheles gambiae Kisumu strain in cone bioassays while PBO bioefficacy will be monitored using pyrethroid resistant strains with overexpressed P450 enzymes in tunnel tests to determine the proportion of efficacious nets (≥ 95% knockdown, ≥ 80% mortality or ≥ 90% blood feeding inhibition in tunnels) at each time point. Nets withdrawn at 12, 24 and 36 months from each country will also be tested in experimental hut trials against wild free-flying pyrethroid resistant Anopheles gambiae sl in Côvè Benin to investigate the superiority of DuraNet® Plus over DuraNet® at each time point under semi field conditions. CONCLUSION: This large-scale multi country trial will provide useful information on the durability of a pyrethroid-PBO net (DuraNet® Plus) in 3 different regions in sub-Saharan Africa. The methods proposed for bioefficacy testing could also contribute towards the development of new standardised guidelines for monitoring the insecticidal efficacy of pyrethroid-PBO nets under operational conditions.

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.

Can the performance of pyrethroid-chlorfenapyr nets be reduced when combined with pyrethroid-piperonyl butoxide (PBO) nets?

BACKGROUND: Pyrethroid-chlorfenapyr (CFP) and pyrethroid-piperonyl butoxide (PBO) nets are being scaled across endemic countries to improve control of malaria transmitted by pyrethroid-resistant mosquitoes. CFP is a pro-insecticide requiring activation by mosquito cytochrome P450 monooxygenase enzymes (P450s) while PBO improves pyrethroid potency by inhibiting the action of these enzymes in pyrethroid-resistant mosquitoes. The inhibitory action of PBO against P450s may thus reduce the efficacy of pyrethroid-CFP nets when applied inside the same household as pyrethroid-PBO nets. METHODS: Two experimental hut trials were performed to evaluate the entomological impact of two different types of pyrethroid-CFP ITN (Interceptor® G2, PermaNet® Dual) when applied alone and in combination with pyrethroid-PBO ITNs (DuraNet® Plus, PermaNet® 3.0) against a pyrethroid-resistant vector population in southern Benin. In both trials, all net types were tested as single and double net treatments. Bioassays were also performed to assess the resistance profile of the vector population at the hut site and investigate interactions between CFP and PBO. RESULTS: The vector population was susceptible to CFP but exhibited a high intensity of pyrethroid resistance that was overcame by PBO pre-exposure. Vector mortality was significantly lower in huts with combinations of pyrethroid-CFP nets plus pyrethroid-PBO nets compared to huts with two pyrethroid-CFP nets (74% vs. 85% for Interceptor® G2 and 57% vs. 83% for PermaNet® Dual, p < 0.001). PBO pre-exposure reduced the toxicity of CFP in bottle bioassays suggesting this effect may be partly attributable to antagonism between CFP and PBO. Higher levels of vector mortality were observed in huts with net combinations that included pyrethroid-CFP nets compared to those that did not and highest mortality was achieved when pyrethroid-CFP nets were applied alone as two nets together (83-85%). CONCLUSIONS: This study shows evidence of a reduced performance of pyrethroid-CFP nets when combined with pyrethroid-PBO ITNs compared to when applied alone and higher efficacy with net combinations that included pyrethroid-CFP nets. These findings suggest that in similar contexts, prioritizing distribution of pyrethroid-CFP nets over other net types would maximize vector control impact.

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.

Comparative efficacy of two pyrethroid-piperonyl butoxide nets (Olyset Plus and PermaNet 3.0) against pyrethroid resistant malaria vectors: a non-inferiority assessment.

BACKGROUND: Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosquitoes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the comparative performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. METHODS: An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization (WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain findings in the experimental huts. RESULTS: With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P < 0.001). After 20 washes, mortality declined significantly with PermaNet 3.0 (41% unwashed vs. 17% after washing P < 0.001), but not with Olyset Plus (28% unwashed vs. 24% after washing P = 0.433); Olyset Plus induced significantly higher mortality than PermaNet 3.0 and Olyset Net after 20 washes. PermaNet 3.0 showed a higher wash retention of PBO compared to Olyset Plus. A non-inferiority analysis performed with data from unwashed and washed nets together using a margin recommended by the WHO, showed that PermaNet 3.0 was non-inferior to Olyset Plus in terms of mosquito mortality (25% with Olyset Plus vs. 27% with PermaNet 3.0, OR = 1.528, 95%CI = 1.02-2.29) but not in reducing mosquito feeding (25% with Olyset Plus vs. 30% with PermaNet 3.0, OR = 1.192, 95%CI = 0.77-1.84). Both pyrethroid-PBO nets were superior to Olyset Net. CONCLUSION: Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mosquito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortality. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa.

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.