Pyrethroid Resistant Anopheles Gambiae Research Articles

Efficacy of Interceptor G2, Royal Guard and PermaNet 3.0 against pyrethroid-resistant Anopheles gambiae s.l. from Za-Kpota, southern Benin: an experimental hut trial

BackgroundThe widespread use of insecticide-treated nets (ITNs) has significantly contributed to the reduction in malaria cases and deaths observed across Africa. Unfortunately, this control strategy is threatened by the rapid spread of pyrethroid resistance in malaria vectors. Dual-active-ingredient insecticidal nets are now available to mitigate the impact of pyrethroid resistance. To facilitate evidence-based decisions regarding product selection in specific use settings, data are needed on the efficacy of these different nets against local mosquito populations.MethodsTwo experimental hut trials were performed in Za-Kpota, southern Benin in 2021 to evaluate the performance of Interceptor G2 (BASF), Royal Guard (Disease Control Technologies) and PermaNet 3.0 (Vestergaard Frandsen), all dual-active-ingredient bednets, in comparison to untreated or standard pyrethroid-treated bednets, against free-flying wild Anopheles gambiae mosquitoes. The performance of some of these next-generation nets was compared to the same type of nets that have been in use for up to 2 years. Mosquitoes collected in the huts were followed up after exposure to assess the sublethal effects of treatments on certain life-history traits.ResultsThe predominant species in the study site was Anopheles gambiae sensu stricto (An. gambiae s.s.). Both Anopheles coluzzii and An. gambiae s.s. were resistant to pyrethroids (deltamethrin susceptibility was restored by piperonyl butoxide pre-exposure). In the experimental hut trials, the highest blood-feeding inhibition (5.56%) was recorded for the Royal Guard net, relative to the standard PermaNet 2.0 net (44.44% inhibition). The highest 72-h mortality rate (90.11%) was recorded for the Interceptor G2 net compared to the PermaNet 2.0 net (56.04%). After exposure, the risk of death of An. gambiae sensu lato (An. gambiae s.l.) was 6.5-fold higher with the Interceptor G2 net and 4.4-fold higher with the PermaNet 3.0 net compared to the respective untreated net. Lower mosquito mortality was recorded with an aged Interceptor G2 net compared to a new Interceptor G2 net. Oviposition rates were lower in mosquitoes collected from huts containing ITNs compared to those of untreated controls. None of the mosquitoes collected from huts equipped with Royal Guard nets laid any eggs.ConclusionsThe Royal Guard and Interceptor G2 nets showed a potential to significantly improve the control of malaria-transmitting vectors. However, the PermaNet 3.0 net remains effective in pyrethroid-resistant areas.Graphical

Bio-efficacy of Olyset® Plus, PermaNet® 3.0 and Interceptor® G2 on pyrethroid-resistant populations of Anopheles gambiae s.l. prior to the June 2023 net distribution campaign in Benin, West Africa

BackgroundThis study investigates the effectiveness of new-generation mosquito nets, like Olyset® Plus and PermaNet® 3.0, and dual-action nets such as Interceptor® G2, against pyrethroid-resistant Anopheles gambiae mosquitoes following the 2023 mass distribution of long-lasting insecticidal nets in Benin.MethodsWe tested wild mosquito populations from six communes in Benin against various pyrethroid (permethrin 0.75%, alphacypermethrin 0.05%, and deltamethrin 0.05%) using WHO tube tests. Additionally, we exposed mosquitoes to chlorfenapyr 100 µg/ml using the CDC bottle bioassay method. A subset of mosquitoes underwent biochemical and PCR tests to check the overexpression of metabolic enzymes and the Kdr L1014F mutation. We evaluated the effectiveness of Olyset® Plus, PermaNet® 3.0, and Interceptor® G2 nets using cone and tunnel tests on both laboratory and field populations of An. gambiae.ResultsOverall, the highest mortality rate was 60% with pyrethroid and 98 to100% with chlorfenapyr. In cone tests, all three types of nets induced mortality rates above 80% in the susceptible laboratory strain of An. gambiae. Notably, Olyset® Plus showed the highest mortality rates for pyrethroid-resistant mosquitoes in cone tests, ranging from 81.03% (95% CI: 68.59–90.13) in Djougou to 96.08% (95% CI: 86.54–99.52) in Akpro-Missérété. PermaNet® 3.0 had variable rates, from 42.5% (95% CI: 27.04–59.11) in Djougou to 58.54% (95% CI: 42.11–73.68) in Porto-Novo. However, revealed good results for Interceptor® G2, with 94% (95% CI: 87.40–97.77) mortality and 89.09% blood sampling inhibition in local populations of An. gambiae. In comparison, Interceptor® had lower rates of 17% (95% CI: 10.23–25.82) and 60%, respectively.ConclusionThese results suggest that tunnel tests are effective for evaluating dual-active ingredient nets. Additionally, Interceptor® G2 and PBO nets like Olyset® Plus could be considered as alternatives against pyrethroid-resistant mosquitoes.

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

BackgroundClothianidin-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.MethodsWe 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.ResultsThe 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.ConclusionsThis 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.Graphical

Insecticidal roof barriers mounted on untreated bed nets can be as effective against Anopheles gambiae as regular insecticide-treated bed nets

Barrier bednets (BBnets), regular bednets with a vertical insecticidal panel to target mosquitoes above the bednet roof, where they are most active, have the potential to improve existing Insecticidal Treated Bednets (ITNs), by reducing the quantity of insecticide required per net, reducing the toxic risks to those using the net, potentially increasing insecticide choice. We evaluated the performance of PermaNet 3.0 (P3) and untreated (Ut) bed nets with and without pyrethroid and piperonyl butoxide roof barriers in killing pyrethroid-resistant and susceptible Anopheles gambiae, simultaneously video-recording mosquito flight tracks. Bioassay results showed that treated roof barriers, particularly the longitudinal P3 barrier (P3L) could be an effective addition to a bed net: P3 + P3L were consistently significantly more effective than the reference P3 bednet while performance of untreated nets could be raised to equal that of the reference P3 following the addition of a P3 barrier. The BBnet’s potential to augment existing bednets and enhance their performance is considered.

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

BackgroundPyrethroid-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.MethodsThe 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.ConclusionThis 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.

Attractive targeted sugar bait: the pyrrole insecticide chlorfenapyr and the anti-malarial pharmaceutical artemether–lumefantrine arrest Plasmodium falciparum development inside wild pyrethroid-resistant Anopheles gambiae s.s. mosquitoes

BackgroundAttractive targeted sugar bait (ATSB) is a novel approach to vector control, offering an alternative mode of insecticide delivery via the insect alimentary canal, with potential to deliver a variety of compounds new to medical entomology and malaria control. Its potential to control mosquitoes was recently demonstrated in major field trials in Africa. The pyrrole chlorfenapyr is an insecticide new to malaria vector control, and through its unique mode of action—disruption of ATP mediated energy transfer in mitochondria—it may have direct action on energy transfer in the flight muscle cells of mosquitoes. It may also have potential to disrupt mitochondrial function in malarial parasites co-existing within the infected mosquito. However, little is known about the impact of such compounds on vector competence in mosquitoes responsible for malaria transmission.MethodsIn this study, ATSBs containing chlorfenapyr insecticide and, as a positive control, the anti-malarial drugs artemether/lumefantrine (A/L) were compared for their effect on Plasmodium falciparum development in wild pyrethroid-resistant Anopheles gambiae sensu stricto (s.s.) and for their capacity to reduce vector competence. Female mosquitoes were exposed to ATSB containing either sublethal dose of chlorfenapyr (CFP: 0.025%) or concentrations of A/L ranging from 0.4/2.4 mg/ml to 2.4/14.4 mg/ml, either shortly before or after taking infective blood meals. The impact of their component compounds on the prevalence and intensity of P. falciparum infection were compared between treatments.ResultsBoth the prevalence and intensity of infection were significantly reduced in mosquitoes exposed to either A/L or chlorfenapyr, compared to unexposed negative control mosquitoes. The A/L dose (2.4/14.4 mg/ml) totally erased P. falciparum parasites: 0% prevalence of infection in female mosquitoes exposed compared to 62% of infection in negative controls (df = 1, χ2 = 31.23 p < 0.001). The dose of chlorfenapyr (0.025%) that killed < 20% females in ATSB showed a reduction in oocyte density of 95% per midgut (0.18/3.43 per midgut).ConclusionThese results are evidence that chlorfenapyr, in addition to its direct killing effect on the vector, has the capacity to block Plasmodium transmission by interfering with oocyte development inside pyrethroid-resistant mosquitoes, and through this dual action may potentiate its impact under field 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.

Sub-lethal exposure to chlorfenapyr reduces the probability of developing Plasmodium falciparum parasites in surviving Anopheles mosquitoes

BackgroundPyrethroid resistance in the key malaria vectors threatens the success of pyrethroid-treated nets. To overcome pyrethroid resistance, Interceptor® G2 (IG2), a ‘first-in-class’ dual insecticidal net that combines alpha-cypermethrin with chlorfenapyr, was developed. Chlorfenapyr is a pro-insecticide, requiring bio-activation by oxidative metabolism within the insect’s mitochondria, constituting a mode of action preventing cross-resistance to pyrethroids. Recent epidemiological trials conducted in Benin and Tanzania confirm IG2’s public health value in areas with pyrethroid-resistant Anopheles mosquitoes. As chlorfenapyr might also interfere with the metabolic mechanism of the Plasmodium parasite, we hypothesised that chlorfenapyr may provide additional transmission-reducing effects even if a mosquito survives a sub-lethal dose.MethodsWe tested the effect of chlorfenapyr netting to reduce Plasmodium falciparum transmission using a modified WHO tunnel test with a dose yielding sub-lethal effects. Pyrethroid-resistant Anopheles gambiae s.s. with L1014F and L1014S knockdown resistance alleles and expression levels of pyrethroid metabolisers CYP6P3, CYP6M2, CYP4G16 and CYP6P1 confirmed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) prior to conducting experiments were exposed to untreated netting and netting treated with 200 mg/m3 chlorfenapyr for 8 h overnight and then fed on gametocytemic blood meals from naturally infected individuals. Prevalence and intensity of oocysts and sporozoites were determined on day 8 and day 16 after feeding.ResultsBoth prevalence and intensity of P. falciparum infection in the surviving mosquitoes were substantially reduced in the chlorfenapyr-exposed mosquitoes compared to untreated nets. The odds ratios in the prevalence of oocysts and sporozoites were 0.33 (95% confidence interval; 95% CI 0.23–0.46) and 0.43 (95% CI 0.25–0.73), respectively, while only the incidence rate ratio for oocysts was 0.30 (95% CI 0.22–0.41).ConclusionWe demonstrated that sub-lethal exposure of pyrethroid-resistant mosquitoes to chlorfenapyr substantially reduces the proportion of infected mosquitoes and the intensity of the P. falciparum infection. This will likely also contribute to the reduction of malaria in communities beyond the direct killing of mosquitoes.Graphical

Efficacy of a ‘lethal house lure’ against Culex quinquefasciatus from Bouaké city, Côte d’Ivoire

BackgroundEave tube technology is a novel method of insecticide application that uses an electrostatic coating system to boost insecticide efficacy against resistant mosquitoes. A series of previous experiments showed encouraging insecticidal effects against malaria vectors. This study was undertaken to assess the effects of the eave tube approach on other Culicidae, in particular Culex quinquefasciatus, under laboratory and semi-field conditions.MethodsLarvae of Cx. quinquefasciatus from Bouaké were collected and reared to adult stage, and World Health Organization (WHO) cylinder tests were performed to determine their resistance status. WHO standard 3-min cone bioassays were conducted using PermaNet 2.0 netting versus eave tube-treated inserts. To assess the transient exposure effect on Cx. quinquefasciatus, eave tube assay utilizing smelly socks as attractant was performed with exposure time of 30 s, 1 min, and 2 min on 10% beta-cyfluthrin-treated inserts. Residual activity of these treated inserts was then monitored over 9 months. Field tests involving release–recapture of Cx. quinquefasciatus within enclosures around experimental huts fitted with windows and untreated or insecticide-treated eave tubes were conducted to determine house entry preference and the impact of tubes on the survival of this species.ResultsBouaké Cx. quinquefasciatus displayed high resistance to three out of four classes of insecticides currently used in public health. After 3 min of exposure in cone tests, 10% beta-cyfluthrin-treated inserts induced 100% mortality in Cx. quinquefasciatus, whereas the long-lasting insecticidal net (LLIN) only killed 4.5%. With reduced exposure time on the eave tube insert, mortality was still 100% after 2 min, 88% after 1 min, and 44% after 30 s. Mortality following 1 h exposure on 10% beta-cyfluthrin-treated insert was > 80% continuously up to 7 months post-treatment. Data suggest that Cx. quinquefasciatus have a stronger preference for entering a house through the eaves than through windows. Beta-cyfluthrin-treated inserts were able to kill 51% of resistant Cx. quinquefasciatus released within the enclosure.ConclusionsEave tubes are a novel method for delivery of insecticide to the house. They attract nuisance host-seeking Cx. quinquefasciatus mosquitoes and are as effective in controlling them as they are against pyrethroid-resistant Anopheles gambiae, despite the high level of resistance Cx. quinquefasciatus have developed.Graphical

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.

Evaluation of Durability as a Function of Fabric Strength and Residual Bio-Efficacy for the Olyset Plus and Interceptor G2 LLINs after 3 Years of Field Use in Tanzania.

Long-lasting insecticidal nets (LLINs) are prone to reduction in insecticide content and physical strength due to repeated washes and usage. The significant loss to these features jeopardizes their protection against bites from malaria vectors. Insecticide washout is attributed to routine use, friction, and washing, while fabric damage is associated with routine use in households. To maintain coverage and cost-effectiveness, nets should maintain optimal bio-efficacy and physical strength for at least 3 years after distribution. In this study, the bio-efficacy and fabric strength of Olyset plus (OP) LLINs and Interceptor G2 (IG2), that were used for 3 years, were assessed in comparison to untreated and new unwashed counterparts. Both IG2 and OP LLINs (unused, laboratory-washed, and 36 months used) were able to induce significant mortality and blood feeding inhibition (BFI) to mosquitoes compared to the untreated nets. Significantly higher mortality was induced by unused IG2 LLIN and OP LLIN compared to their 36-month-old counterparts against both pyrethroid resistant and susceptible Anopheles gambiae sensu strito. The physical strength of the IG2 LLIN was higher than that of the Olyset Plus LLIN with a decreasing trend from unwashed, laboratory-washed to community usage (36 months old). Malaria control programs should consider bio-efficacy and physical integrity prior to an LLINs' procurement and replacement plan.

Selection for insecticide resistance can promote Plasmodium falciparum infection in Anopheles.

Insecticide resistance is under strong selective pressure in Anopheles mosquitoes due to widespread usage of insecticides in vector control strategies. Resistance mechanisms likely cause changes that profoundly affect mosquito physiology, yet it remains poorly understood how selective pressures imposed by insecticides may alter the ability of the mosquito to host and transmit a Plasmodium infection. From pyrethroid-resistant field-derived Anopheles gambiae s.l. mosquitoes, we established resistant (RES) and susceptible (SUS) colonies by either selection for, or loss of insecticide resistance. We show increased oocyst intensity and growth rate as well as increased sporozoite prevalence and intensity in RES compared to SUS females infected with Plasmodium falciparum. The increase in infection intensity in RES females was not associated with the presence of the kdrL1014F mutation and was not impacted by inhibition of Cytochrome P450s. The lipid transporter lipophorin (Lp), which was upregulated in RES compared to SUS, was at least partly implicated in the increased intensity of P. falciparum but not directly involved in the insecticide resistance phenotype. Interestingly, we observed that although P. falciparum infections were not affected when RES females were exposed to permethrin, these females had decreased lipid abundance in the fat body following exposure, pointing to a possible role for lipid mobilization in response to damage caused by insecticide challenge. The finding that selection for insecticide resistance can increase P. falciparum infection intensities and growth rate reinforces the need to assess the overall impact on malaria transmission dynamics caused by selective pressures mosquitoes experience during repeated insecticide challenge.

Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking

BackgroundThe success of insecticide treated bed nets (ITNs) for malaria vector control in Africa relies on the behaviour of various species of Anopheles. Previous research has described mosquito behavioural alterations resulting from widespread ITN coverage, which could result in a decrease in net efficacy. Here, behaviours were compared including timings of net contact, willingness to refeed and longevity post-exposure to two next-generation nets, PermaNet® 3.0 (P3 net) and Interceptor® G2 (IG2 net) in comparison with a standard pyrethroid-only net (Olyset Net™ (OL net)) and an untreated net.MethodsSusceptible and resistant Anopheles gambiae mosquitoes were exposed to the nets with a human volunteer host in a room-scale assay. Mosquito movements were tracked for 2 h using an infrared video system, collecting flight trajectory, spatial position and net contact data. Post-assay, mosquitoes were monitored for a range of sublethal insecticide effects.ResultsMosquito net contact was focused predominantly on the roof for all four bed nets. A steep decay in activity was observed for both susceptible strains when P3 net and OL net were present and with IG2 net for one of the two susceptible strains. Total mosquito activity was higher around untreated nets than ITNs. There was no difference in total activity, the number, or duration, of net contact, between any mosquito strain, with similar behaviours recorded in susceptible and resistant strains at all ITNs. OL net, P3 net and IG2 net all killed over 90% of susceptible mosquitoes 24 h after exposure, but this effect was not seen with resistant mosquitoes where mortality ranged from 16 to 72%. All treated nets reduced the willingness of resistant strains to re-feed when offered blood 1-h post-exposure, with a more pronounced effect seen with P3 net and OL net than IG2 net.ConclusionThese are the first results to provide an in-depth description of the behaviour of susceptible and resistant Anopheles gambiae strains around next-generation bed nets using a room-scale tracking system to capture multiple behaviours. These results indicate that there is no major difference in behavioural responses between mosquito strains of differing pyrethroid susceptibility when exposed to these new ITNs under the experimental conditions used.

Exploring alternative insecticide delivery options in a “lethal house lure” for malaria vector control

The In2Care EaveTube is a house modification designed to block and kill malaria mosquitoes using an electrostatic netting treated with insecticide powder. A previous study demonstrated prolonged duration of effective action of insecticide-treated electrostatic netting in a semi-field setting. As part of a cluster randomized controlled trial (CRT) of the EaveTube intervention in Côte d’Ivoire, we investigated the residual efficacy of a pyrethroid insecticide deployed in EaveTubes under village conditions of use. We also explored the scope of using existing malaria control technologies including LLINs and IRS as alternative methods to deliver insecticides in the lethal house lure. The efficacy of beta-cyfluthrin was monitored over time using the “eave tube bioassay” method. Mortality of beta-cyfluthrin exposed pyrethroid resistant Anopheles gambiae mosquitoes was > 80% after 4 months. The impact (mosquito mortality) of PVC tubes coated with pirimiphos methyl was similar to that of beta-cyfluthrin treated insert (66.8 vs. 62.8%) in release-recapture experiments in experimental huts. Efficacy was significantly lower with all the LLINs tested; however, the roof of PermaNet 3.0 induced significantly higher mosquito mortality (50.4%) compared to Olyset Plus (25.9%) and Interceptor G2 (21.6%) LLINs. The efficacy of the alternative delivery methods was short-lived with mortality decreasing below 50% within 2 months in residual activity bioassays. None of the products tested appeared superior to the powder treatments. Further research is therefore required to identify suitable insecticide delivery options in EaveTube for malaria vector control.

Small-scale field evaluation of PermaNet® Dual (a long-lasting net coated with a mixture of chlorfenapyr and deltamethrin) against pyrethroid-resistant Anopheles gambiae mosquitoes from Tiassalé, Côte d’Ivoire

BackgroundDue to the rapid expansion of pyrethroid-resistance in malaria vectors in Africa, Global Plan for Insecticide Resistance Management (GPIRM) has recommended the development of long-lasting insecticidal nets (LLINs), containing insecticide mixtures of active ingredients with different modes of action to mitigate resistance and improve LLIN efficacy. This good laboratory practice (GLP) study evaluated the efficacy of the chlorfenapyr and deltamethrin-coated PermaNet® Dual, in comparison with the deltamethrin and synergist piperonyl butoxide (PBO)-treated PermaNet® 3.0 and the deltamethrin-coated PermaNet® 2.0, against wild free-flying pyrethroid-resistant Anopheles gambiae sensu lato (s.l.), in experimental huts in Tiassalé, Côte d’Ivoire (West Africa).MethodsPermaNet® Dual, PermaNet® 3.0 and PermaNet® 2.0, unwashed and washed (20 washes), were tested against free-flying pyrethroid-resistant An. gambiae s.l. in the experimental huts in Tiassalé, Côte d’Ivoire from March to August 2020. Complementary laboratory cone bioassays (daytime and 3-min exposure) and tunnel tests (nightly and 15-h exposure) were performed against pyrethroid-susceptible An. gambiae sensu stricto (s.s.) (Kisumu strain) and pyrethroid-resistant An. gambiae s.l. (Tiassalé strain).ResultsPermaNet® Dual demonstrated significantly improved efficacy, compared to PermaNet® 3.0 and PermaNet® 2.0, against the pyrethroid-resistant An. gambiae s.l. Indeed, the experimental hut trial data showed that the mortality and blood-feeding inhibition in the wild pyrethroid-resistant An. gambiae s.l. were overall significantly higher with PermaNet® Dual compared with PermaNet® 3.0 and PermaNet® 2.0, for both unwashed and washed samples. The mortality with unwashed and washed samples were 93.6 ± 0.2% and 83.2 ± 0.9% for PermaNet® Dual, 37.5 ± 2.9% and 14.4 ± 3.9% for PermaNet® 3.0, and 7.4 ± 5.1% and 11.7 ± 3.4% for PermaNet® 2.0, respectively. Moreover, unwashed and washed samples produced the respective percentage blood-feeding inhibition of 41.4 ± 6.9% and 43.7 ± 4.8% with PermaNet® Dual, 51.0 ± 5.7% and 9.8 ± 3.6% with PermaNet® 3.0, and 12.8 ± 4.3% and − 13.0 ± 3.6% with PermaNet® 2.0. Overall, PermaNet® Dual also induced higher or similar deterrence, exophily and personal protection when compared with the standard PermaNet® 3.0 and PermaNet® 2.0 reference nets, with both unwashed and washed net samples. In contrast to cone bioassays, tunnel tests predicted the efficacy of PermaNet® Dual seen in the current experimental hut trial.ConclusionThe deltamethrin-chlorfenapyr-coated PermaNet® Dual induced a high efficacy and performed better than the deltamethrin-PBO PermaNet® 3.0 and the deltamethrin-only PermaNet® 2.0, testing both unwashed and 20 times washed samples against the pyrethroid-susceptible and resistant strains of An. gambiae s.l. The inclusion of chlorfenapyr with deltamethrin in PermaNet® Dual net greatly improved protection and control of pyrethroid-resistant An. gambiae populations. PermaNet® Dual thus represents a promising tool, with a high potential to reduce malaria transmission and provide community protection in areas compromised by mosquito vector resistance to pyrethroids.

A non-inferiority and GLP-compliant study of broflanilide IRS (VECTRON™ T500), a novel meta-diamide insecticide against Anopheles arabiensis

Management of insecticide resistance in vector control requires development and evaluation of active ingredients (AIs) with new modes of action. VECTRON™ T500 is a wettable powder formulation used for Indoor Residual Spraying (IRS) containing 50% of broflanilide as an AI. This study evaluated the efficacy of VECTRON™ T500 sprayed on blocks of different substrates (concrete, mud and plywood) against pyrethroid susceptible and resistant Anopheles gambiae sensu stricto (s.s.) strains, and wild An. arabiensis. It also assessed the efficacy of VECTRON™ T500 in experimental huts plastered with mud and concrete against wild free-flying An. arabiensis; and non-inferiority to a World Health Organization listed indoor residual spraying product Actellic® 300CS in terms of mortality in Moshi, Tanzania.Monthly cone bioassays on blocks and in experimental huts (against pyrethroid susceptible and resistant An. gambiae s.s.) were conducted over a 12-month period after spraying of VECTRON™ T500 and Actellic® CS300. Collections of wild free-flying An. arabiensis were also done in the sprayed huts. The main outcome for cone bioassays was mortality while for the wild hut trial collections, it was mortality and blood feeding inhibition. Grouped logistic regressions with random effects were used to analyse all dichotomous outcome variables from wild collections.The results showed residual efficacy of VECTRON™ T500 of at least 80% mortality was longest on concrete, followed by plywood and then mud substrates for all mosquito strains. Furthermore, VECTRON™ T500 significantly increased the likelihood of mortality (OR:&amp;gt; 1.37, P&amp;lt;0.001) in wild collections of An. arabiensis compared to Actellic® 300CS. Blood feeding was not significantly different in the wild collection of An. arabiensis between VECTRON™ T500 and Actellic® 300CS arms.These results show that VECTRON™ T500 is efficacious against pyrethroid-resistant An. gambiae s.s. and non-inferior to Actellic® 300CS. Therefore, it should be an important addition to the current arsenal of insecticides used for insecticide resistance management and vector control.

Laboratory and semi-field efficacy evaluation of permethrin–piperonyl butoxide treated blankets against pyrethroid-resistant malaria vectors

To control pyrethroid-resistant malaria vectors, Indoor Residual Spraying (IRS) and Long-Lasting Insecticidal Nets (LLINs) that include additional ingredients to pyrethroid are being developed. Same progress needs to be made to the pyrethroid-treated blankets, which are more compatible with shelter structures found in emergency settings such as displaced populations. In the current study, efficacy of blankets treated with permethrin and piperonyl butoxide (PBO) was evaluated against pyrethroid-resistant Anopheles gambiae sensu stricto. Efficacy was compared with that of Olyset LLIN, Olyset Plus LLIN and untreated blanket in terms of mortality and blood-feeding inhibition against pyrethroid-resistant Anopheles gambiae mosquitoes. The current study indicates that, in emergency shelters such as migrant and refugee camps where LLINs cannot be used, PBO–permethrin blankets may provide protection against resistant mosquitoes if widely used. No side effects related to the use of the treated blankets were reported from the participants. These results need validation in a large-scale field trial to assess the epidemiological impact of the intervention, durability and acceptability of this new vector control strategy for malaria vector control.

VECTRON™ T500, a new broflanilide insecticide for indoor residual spraying, provides prolonged control of pyrethroid-resistant malaria vectors

BackgroundBroflanilide 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.MethodsVECTRON™ 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.ResultsThe 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.ConclusionVECTRON™ 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.

Reduced performance of community bednets against pyrethroid-resistant Anopheles funestus and Anopheles gambiae, major malaria vectors in Cameroon

BackgroundLong-lasting insecticidal nets (LLINs) are a vital tool in the fight against malaria vectors. However, their efficacy in the field can be impacted by several factors, including patterns of usage, net age, mosquito resistance and the delayed mortality effect, all of which could influence malaria transmission. We have investigated the effectiveness of the various brands of LLINs available in markets and households in Cameroon on pyrethroid-resistant mosquitoes and assessed their post-exposure effect.MethodsFollowing quality control assessment on a susceptible laboratory mosquito strain, we evaluated the immediate and delayed mortality effects of exposure to LLINs (both newly bough LLINst and used ones collected from households in Elende village, Cameroon, in 2019) using standard WHO cone tests on Anopheles gambiae and Anopheles funestus populations collected from the Centre region of Cameroon. Alive female mosquitoes were genotyped for various resistance markers at different time points post-exposure to evaluate the impact of insecticide resistance on the efficacy of bednets.ResultsThe laboratory-susceptible strain experienced high mortality rates when exposed to all pyrethroid-only brands of purchased nets (Olyset® Net, Super Net, PermaNet® 2.0, Yorkool®, Royal Sentry®) (Mean±SEM: 68.66 ± 8.35% to 93.33 ± 2.90%). However, low mortality was observed among wild An. funestus mosquitoes exposed to the bednets (0 ± 0 to 28 ± 6.7%), indicating a reduced performance of these nets against field mosquitoes. Bednets collected from households also showed reduced efficacy on the laboratory strain (mortality: 19–66%), as well as displaying a significant loss of efficacy against the local wild strains (mortality: 0 ± 0% to 4 ± 2.6% for An. gambiae sensu lato and 0 ± 0% to 8 ± 3.2% for An. funestus). However, compared to the unexposed group, mosquitoes exposed to bednets showed a significantly reduced longevity, indicating that the efficacy of these nets was not completely lost. Mosquitoes with the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance showed greater longevity after exposure to the Olyset net than their susceptible counterparts, indicating the impact of resistance on bednet efficacy and delayed mortality.ConclusionThese findings show that although standard bednets drastically lose their efficacy against pyrethroid-resistant field mosquitoes, they still are able to induce delayed mortality in exposed populations. The results of this study also provide evidence of the actual impact of resistance on the quality and efficacy of LLINs in use in the community, with mosquitoes carrying the CYP6P9a-RR and L119F-GSTe2 mutations conferring pyrethroid resistance living longer than their susceptible counterparts. These results highlight the need to use new-generation nets that do not rely solely on pyrethroids.Graphical

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.