Efficacy of pyriproxyfen-pyrethroid long-lasting insecticidal nets (LLINs) and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs for malaria control in Benin: a cluster-randomised, superiority trial.

BACKGROUND: New classes of long-lasting insecticidal nets (LLINs) combining mixtures of insecticides with different modes of action could put malaria control back on track after rebounds in transmission across sub-Saharan Africa. We evaluated the relative efficacy of pyriproxyfen-pyrethroid LLINs and chlorfenapyr-pyrethroid LLINs compared with standard LLINs against malaria transmission in an area of high pyrethroid resistance in Benin. METHODS: We conducted a cluster-randomised, superiority trial in Zou Department, Benin. Clusters were villages or groups of villages with a minimum of 100 houses. We used restricted randomisation to randomly assign 60 clusters to one of three LLIN groups (1:1:1): to receive nets containing either pyriproxyfen and alpha-cypermethrin (pyrethroid), chlorfenapyr and alpha-cypermethrin, or alpha-cypermethrin only (reference). Households received one LLIN for every two people. The field team, laboratory staff, analyses team, and community members were masked to the group allocation. The primary outcome was malaria case incidence measured over 2 years after net distribution in a cohort of children aged 6 months-10 years, in the intention-to-treat population. This study is ongoing and is registered with ClinicalTrials.gov, NCT03931473. FINDINGS: Between May 23 and June 24, 2019, 53 854 households and 216 289 inhabitants were accounted for in the initial census and included in the study. Between March 19 and 22, 2020, 115 323 LLINs were distributed to 54 030 households in an updated census. A cross-sectional survey showed that study LLIN usage was highest at 9 months after distribution (5532 [76·8%] of 7206 participants), but decreased by 24 months (4032 [60·6%] of 6654). Mean malaria incidence over 2 years after LLIN distribution was 1·03 cases per child-year (95% CI 0·96-1·09) in the pyrethroid-only LLIN reference group, 0·84 cases per child-year (0·78-0·90) in the pyriproxyfen-pyrethroid LLIN group (hazard ratio [HR] 0·86, 95% CI 0·65-1·14; p=0·28), and 0·56 cases per child-year (0·51-0·61) in the chlorfenapyr-pyrethroid LLIN group (HR 0·54, 95% CI 0·42-0·70; p<0·0001). INTERPRETATION: Over 2 years, chlorfenapyr-pyrethroid LLINs provided greater protection from malaria than pyrethroid-only LLINs in an area with pyrethroid-resistant mosquitoes. Pyriproxyfen-pyrethroid LLINs conferred protection similar to pyrethroid-only LLINs. These findings provide crucial second-trial evidence to enable WHO to make policy recommendations on these new LLIN classes. This study confirms the importance of chlorfenapyr as an LLIN treatment to control malaria in areas with pyrethroid-resistant vectors. However, an arsenal of new active ingredients is required for successful long-term resistance management, and additional innovations, including pyriproxyfen, need to be further investigated for effective vector control strategies. FUNDING: UNITAID, The Global Fund.

Identifying suitable methods for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors: a comparison of the oviposition and ovary dissection methods.

BACKGROUND: Nets containing pyriproxyfen, an insect growth regulator that sterilizes adult mosquitoes, have become available for malaria control. Suitable methods for investigating vector susceptibility to pyriproxyfen and evaluating its efficacy on nets need to be identified. The sterilizing effects of pyriproxyfen on adult malaria vectors can be assessed by measuring oviposition or by dissecting mosquito ovaries to determine damage by pyriproxyfen (ovary dissection). METHOD: Laboratory bioassays were performed to compare the oviposition and ovary dissection methods for monitoring susceptibility to pyriproxyfen in wild malaria vectors using WHO bottle bioassays and for evaluating its efficacy on nets in cone bioassays. Blood-fed mosquitoes of susceptible and pyrethroid-resistant strains of Anopheles gambiae sensu lato were exposed to pyriproxyfen-treated bottles (100 µg and 200 µg) and to unwashed and washed pieces of a pyriproxyfen long-lasting net in cone bioassays. Survivors were assessed for the sterilizing effects of pyriproxyfen using both methods. The methods were compared in terms of their reliability, sensitivity, specificity, resources (cost and time) required and perceived difficulties by trained laboratory technicians. RESULTS: The total number of An. gambiae s.l. mosquitoes assessed for the sterilizing effects of pyriproxyfen were 1745 for the oviposition method and 1698 for the ovary dissection method. Fertility rates of control unexposed mosquitoes were significantly higher with ovary dissection compared to oviposition in both bottle bioassays (99-100% vs. 34-59%, P < 0.05) and cone bioassays (99-100% vs. 18-33%, P < 0.001). Oviposition rates of control unexposed mosquitoes were lower with wild pyrethroid-resistant An. gambiae s.l. Cové, compared to the laboratory-maintained reference susceptible An gambiae sensu stricto Kisumu (18-34% vs. 58-76%, P < 0.05). Sterilization rates of the Kisumu strain in bottle bioassays with the pyriproxyfen diagnostic dose (100 µg) were suboptimal with the oviposition method (90%) but showed full susceptibility with ovary dissection (99%). Wild pyrethroid-resistant Cové mosquitoes were fully susceptible to pyriproxyfen in bottle bioassays using ovary dissection (> 99%), but not with the oviposition method (69%). Both methods showed similar levels of sensitivity (89-98% vs. 89-100%). Specificity was substantially higher with ovary dissection compared to the oviposition method in both bottle bioassays (99-100% vs. 34-48%) and cone tests (100% vs.18-76%). Ovary dissection was also more sensitive for detecting the residual activity of pyriproxyfen in a washed net compared to oviposition. The oviposition method though cheaper, was less reliable and more time-consuming. Laboratory technicians preferred ovary dissection mostly due to its reliability. CONCLUSION: The ovary dissection method was more accurate, more reliable and more efficient compared to the oviposition method for evaluating the sterilizing effects of pyriproxyfen on adult malaria vectors in susceptibility bioassays and for evaluating the efficacy of pyriproxyfen-treated nets.

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.

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.

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.

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.

Assessing the efficacy of two dual-active ingredients long-lasting insecticidal nets for the control of malaria transmitted by pyrethroid-resistant vectors in Benin: study protocol for a three-arm, single-blinded, parallel, cluster-randomized controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are currently the primary method of malaria control in sub-Saharan Africa and have contributed to a significant reduction in malaria burden over the past 15 years. However, this progress is threatened by the wide-scale selection of insecticide-resistant malaria vectors. It is, therefore, important to accelerate the generation of evidence for new classes of LLINs. METHODS: This protocol presents a three-arm superiority, single-blinded, cluster randomized controlled trial to evaluate the impact of 2 novel dual-active ingredient LLINs on epidemiological and entomological outcomes in Benin, a malaria-endemic area with highly pyrethroid-resistant vector populations. The study arms consist of (i) Royal Guard® LLIN, a net combining a pyrethroid (alpha-cypermethrin) plus an insect growth regulator (pyriproxyfen), which in the adult female is known to disrupt reproduction and egg fertility; (ii) Interceptor G2® LLIN, a net incorporating two adulticides (alpha-cypermethrin and chlorfenapyr) with different modes of action; and (iii) the control arm, Interceptor® LLIN, a pyrethroid (alpha-cypermethrin) only LLIN. In all arms, one net for every 2 people will be distributed to each household. Sixty clusters were identified and randomised 1:1:1 to each study arm. The primary outcome is malaria case incidence measured over 24 months through active case detection in a cohort of 25 children aged 6 months to 10 years, randomly selected from each cluster. Secondary outcomes include 1) malaria infection prevalence (all ages) and prevalence of moderate to severe anaemia in children under 5 years old, measured at 6 and 18 months post-intervention; 2) entomological indices measured every 3 months using human landing catches over 24 months. Insecticide resistance intensity will also be monitored over the study period. DISCUSSION: This study is the second cluster randomised controlled trial to evaluate the efficacy of these next-generation LLINs to control malaria transmitted by insecticide-resistant mosquitoes. The results of this study will form part of the WHO evidence-based review to support potential public health recommendations of these nets and shape malaria control strategies of sub-Saharan Africa for the next decade. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03931473 , registered on 30 April 2019.

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.

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.

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.

The activity of the pyrrole insecticide chlorfenapyr in mosquito bioassay: towards a more rational testing and screening of non-neurotoxic insecticides for malaria vector control.

BACKGROUND: 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. METHODS: The efficacy of WHO recommended cone, cylinder and tunnel tests was compared for pyrethroids and chlorfenapyr. To establish bioassay exposure times predictive of insecticide-treated 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. RESULTS: 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. CONCLUSIONS: Testing according to current WHO guidelines is not suitable for certain types of non-neurotoxic 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 night-active anophelines is the most reliable bioassay for identifying the toxicity of novel insecticides.

Insecticide-treated net wall hangings for malaria vector control: an experimental hut study in north-eastern Tanzania.

BACKGROUND: Alternative long-lasting, practical and effective tools for applying insecticides on home walls against malaria vectors need to be developed. The use of wall hangings made from netting on interior walls for aesthetic purposes is a common practice in rural communities. Insecticide-treated net wall hangings can be produced in a long-lasting format and used in an approach that simulates indoor residual spraying (IRS). METHODS: The efficacy of net wall hangings (NWH) treated with the residual organophosphate insecticide, pirimiphos methyl (1 g/sq m), was evaluated in experimental huts against malaria vectors in Muheza, Tanzania. To determine the optimum level of wall coverage required, NWH were tested on ceiling only, two walls, four walls, or four walls plus ceiling. Comparison was made with deltamethrin-treated NWH on two walls. RESULTS: Pirimiphos methyl (p-methyl)-treated NWH (on two walls) killed significantly higher proportions of anophelines (92% of Anopheles gambiae and 79% of Anopheles funestus) than the deltamethrin-treated NWH (15% of An. gambiae and 17% of An. funestus) (P<0.001). WHO susceptibility tests showed that the local vector population was susceptible to the organophosphates but resistant to pyrethroids. Mortality rates were significantly higher in huts with p-methyl NWH on two walls (92% for An. gambiae and 79% for An. funestus) than on ceiling only (61% for An. gambiae and 62% for An. funestus, P<0.05). There was no improvement in mortality when wall coverage with p-methyl NWH increased beyond two walls. Blood-feeding rates with p-methyl NWH were generally high across all the treatments (52-77%) and did not differ significantly from the control (64-67%). There was no evidence of reduced blood-feeding or increased exiting with increase in wall coverage with p-methyl NWH. CONCLUSIONS: Net wall hangings are an effective means of delivering insecticides in the domestic environment against malaria vectors. They could be more practical and acceptable than IRS thus showing enormous potential for malaria vector control. Appropriate binding or incorporation technology needs to be developed to enable the production of p-methyl NWH with residual activity lasting over a number of years.

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 Anopheles gambiae in Côte d'Ivoire: an experimental hut trial.

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

Laboratory evaluation of the contact irritancy of a clothianidin solo formulation vs. clothianidin-deltamethrin mixture formulations for indoor residual spraying against pyrethroid-resistant Anopheles gambiae sensu lato.

BACKGROUND: Clothianidin-based indoor residual spraying (IRS) formulations have become available for malaria control as either solo formulations of clothianidin or a mixture of clothianidin with the pyrethroid deltamethrin. While both formulations have been successfully used for malaria control, studies investigating the effect of the pyrethroid in IRS mixtures may help improve our understanding for development of future IRS products. It has been speculated that the irritant effect of the pyrethroid in the mixture formulation may result in shorter mosquito contact times with the treated walls potentially leading to a lower impact. METHODS: We compared contact irritancy expressed as the number of mosquito take-offs from cement surfaces treated with an IRS formulation containing clothianidin alone (SumiShield® 50WG) to clothianidin-deltamethrin mixture IRS formulations against pyrethroid-resistant Anopheles gambiae sensu lato under controlled laboratory conditions using a modified version of the World Health Organisation cone bioassay. To control for the pyrethroid, comparison was made with a deltamethrin-only formulation. Both commercial and generic non-commercial mixture formulations of clothianidin and deltamethrin were tested. RESULTS: The clothianidin solo formulation did not show significant contact irritancy relative to the untreated control (3.5 take-offs vs. 3.1 take-offs, p = 0.614) while all deltamethrin-containing IRS induced significant irritant effects. The number of take-offs compared to the clothianidin solo formulation (3.5) was significantly higher with the commercial clothianidin-deltamethrin mixture (6.1, p = 0.001), generic clothianidin-deltamethrin mixture (7.0, p < 0.001), and deltamethrin-only (8.2, p < 0.001) formulations. The commercial clothianidin-deltamethrin mixture induced similar contact irritancy as the generic clothianidin-deltamethrin mixture (6.1 take-offs vs. 7.0 take-offs, p = 0.263) and deltamethrin-only IRS (6.1 take-offs vs. 8.2, p = 0.071), showing that the irritant effect in the mixture was attributable to its deltamethrin component. CONCLUSIONS: This study provides evidence that the enhanced contact irritancy of the pyrethroid in clothianidin-deltamethrin IRS mixtures can shorten mosquito contact times with treated walls compared to the clothianidin solo formulation. Further trials are needed to directly compare the efficacy of these formulation types under field conditions and establish the impact of this enhanced contact irritancy on the performance of IRS mixture formulations containing pyrethroids.

Efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr nets on entomological indicators of malaria transmission: third year of a randomised controlled trial in Benin.

The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46-1.88), p = 0.8527; and RR 1.10 (95% CI 0.44-2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37-1.48), p = 0.3946; and RR 1.00 (95% CI 0.40-2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Effectiveness of pyriproxyfen-pyrethroid and chlorfenapyr-pyrethroid long-lasting insecticidal nets (LLINs) compared with pyrethroid-only LLINs for malaria control in the third year post-distribution: a secondary analysis of a cluster-randomised controlled trial in Benin.

BACKGROUND: Malaria continues to kill approximately 650 000 people each year. There is evidence that some second-generation insecticide-treated nets, which combine insecticide formulations with different modes of action, are protective against malaria while the nets are new; however, evidence for their impact over 3 years is scarce. In this study, we report the third-year results of a cluster-randomised controlled trial assessing the long-term effectiveness of dual-active ingredient long-lasting insecticidal nets (LLINs). METHODS: This is a secondary analysis of a cluster-randomised controlled trial, carried out between May 23, 2019, and April 30, 2023, in southern Benin. Restricted randomisation was used to assign 60 clusters (villages or groups of villages with a minimum of 100 households) to the three study groups (1:1:1) to evaluate the efficacy of pyriproxyfen-pyrethroid LLINs and chlorfenapyr-pyrethroid LLINs compared with pyrethroid-only LLINs (reference) against malaria transmission. The study staff and communities were masked to the group allocation. The primary outcome was malaria incidence measured over the third year after LLIN distribution, in a cohort of children aged 6 months to 9 years at the time of enrolment, in the intention-to-treat population. Here, we present the data of the third year post-LLIN distribution. The trial was registered with ClinicalTrials.gov, NCT03931473. FINDINGS: Study net use declined over the 3 years and was consistently lowest in the pyriproxyfen-pyrethroid LLIN group (at 36 months: 889 [39·4%] of 2257 participants vs 1278 [52·2%] of 2450 participants for the chlorfenapyr-pyrethroid LLIN group and 1400 [57·6%] of 2430 participants for the pyrethroid-only LLIN group). The cohort of children for the third year of follow-up (600 per group) were enrolled between April 9 and 30, 2022. Mean malaria incidence during the third year after distribution was 1·19 cases per child-year (95% CI 1·09-1·29) in the pyrethroid-only LLIN reference group, 1·21 cases per child-year (1·12-1·31) in the pyriproxyfen-pyrethroid LLIN group (hazard ratio [HR] 1·02, 95% CI 0·71-1·44; p=0·92), and 0·96 cases per child-year (0·88-1·05) in the chlorfenapyr-pyrethroid LLIN group (HR 0·80, 0·56-1·17; p=0·25). No adverse events related to study nets were reported by participants. INTERPRETATION: During the third year, as was also observed during the first 2 years, the pyriproxyfen-pyrethroid LLIN group did not have superior protection against malaria cases compared with the standard LLIN group. In the third year, people living in the chlorfenapyr-pyrethroid LLIN group no longer benefited from greater protection against malaria cases and infections than those living in the pyrethroid-only LLIN group. This was probably influenced by lower study net use than previous years and the declining concentration of partner insecticides in the nets. FUNDING: UNITAID, The Global Fund. TRANSLATION: For the French translation of the abstract see Supplementary Materials section.

The impact of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr long-lasting insecticidal nets on density of primary malaria vectors Anopheles gambiae s.s. and Anopheles coluzzii in Benin: a secondary analysis of a cluster randomised controlled trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. METHODS: Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). RESULTS: Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (< 1%) of hybrids (An. gambiae/coluzzii). There was no evidence of a significant reduction in vector density indoors in either primary vector species [An. coluzzii: DR (density ratio) = 0.62 (95% CI 0.21-1.77), p = 0.3683 for the pyrethroid-pyriproxyfen LLIN and DR = 0.56 (95% CI 0.19-1.62), p = 0.2866 for the pyrethroid-chlorfenapyr LLIN, An. gambiae s.s.: DR = 0.52 (95% CI 0.18-1.46), p = 0.2192 for the pyrethroid-pyriproxyfen LLIN and DR = 0.53 (95% CI 0.19-1.46), p = 0.2222 for the pyrethroid-chlorfenapyr]. The same trend was observed outdoors. Parity rates of An. gambiae s.l. were also similar across study arms. CONCLUSIONS: Compared with pyrethroid-only LLINs, pyrethroid-chlorfenapyr LLINs and pyrethroid-pyriproxyfen LLINs performed similarly against the two primary mosquito species An. gambiae s.s. and An. coluzzii in Benin.

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.

PermaNet Dual, a new deltamethrin-chlorfenapyr mixture net, shows improved efficacy against pyrethroid-resistant Anopheles gambiae sensu lato in southern Benin.

Pyrethroid-chlorfenapyr nets have demonstrated improved entomological and epidemiological impact in trials across Africa. This is driving increased demand for this novel net class in malaria-endemic countries. PermaNet Dual is a new deltamethrin-chlorfenapyr net developed by Vestergaard Sàrl to provide more options to malaria control programmes. We performed an experimental hut trial to evaluate the efficacy of PermaNet Dual against wild, free-flying pyrethroid-resistant Anopheles gambiae sensu lato in Covè, Benin. PermaNet Dual induced superior levels of mosquito mortality compared to a pyrethroid-only net and a pyrethroid-piperonyl butoxide net both when unwashed (77% with PermaNet Dual vs. 23% with PermaNet 2.0 and 56% with PermaNet 3.0, p < 0.001) and after 20 standardised washes (75% with PermaNet Dual vs. 14% with PermaNet 2.0 and 30% with PermaNet 3.0, p < 0.001). Using a provisional non-inferiority margin defined by the World Health Organisation, PermaNet Dual was also non-inferior to a pyrethroid-chlorfenapyr net that has demonstrated improved public health value (Interceptor G2), for vector mortality (79% vs. 76%, OR = 0.878, 95% CIs 0.719-1.073) but not for blood-feeding protection (35% vs. 26%, OR = 1.424, 95% CIs 1.177-1.723). PermaNet Dual presents an additional option of this highly effective net class for improved control of malaria transmitted by pyrethroid-resistant mosquitoes.

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.