High efficacy of chlorfenapyr-based net Interceptor■G2 against pyrethroid-resistant malaria vectors from Cameroon

Background The increasing reports of resistance to pyrethroid insecticides associated with reduced efficacy of pyrethroid-only interventions highlight the urgency of introducing new non-pyrethroid-only control tools.Here,we investigated the performance of piperonyl-butoxide(PBO)-pyrethroid[Permanet 3.0(P3.0)]and dual active ingredients(AI)nets[Interceptor G2(IG2):containing pyrethroids and chlorfenapyr and Royal Guard(RG):containing pyrethroids and pyriproxyfen]compared to pyrethroid-only net Royal Sentry(RS)against pyrethroid-resistant malaria vectors in Cameroon.Methods The efficacy of these tools was firstly evaluated onAnopheles gambiae s.l.andAnopheles funestus s.l.from Gounougou,Mibellon,Mangoum,Nkolondom,and Elende using cone/tunnel assays.In addition,experimental hut trials(EHT)were performed to evaluate the performance of unwashed and 20 times washed nets in semi-field conditions.Furthermore,pyrethroid-resistant markers were genotyped in dead vs alive,blood-fed vs unfed mosquitoes after exposure to the nets to evaluate the impact of these markers on net performance.The XLSTAT software was used to calculate the various entomological outcomes and the Chi-square test was used to compare the efficacy of various nets.The odds ratio and Fisher exact test were then used to establish the statistical significance of any association between insecticide resistance markers and bed net efficacy.Results Interceptor G2 was the most effective net against wild pyrethroid-resistantAn.funestus followed by Permanet 3.0.In EHT,this net induced up to 87.8%mortality[95%confidence interval(CI):83.5-92.1%)and 55.6%(95%CI:48.5-62.7%)after 20 washes whilst unwashed pyrethroid-only net(Royal Sentry)killed just 18.2%(95%CI:13.4-22.9%)of host-seekingAn.funestus.The unwashed Permanet 3.0 killed up to 53.8%(95%CI:44.3-63.4%)of field-resistant mosquitoes and 47.2%(95%CI:37.7-56.7%)when washed 20 times,and the Royal Guard 13.2%(95%CI:9.0-17.3%)for unwashed net and 8.5%(95%CI:5.7-11.4%)for the 20 washed net.Interceptor G2,Permanet 3.0,and Royal Guard provided better personal protection(blood-feeding inhibition 66.2%,77.8%,and 92.8%,respectively)compared to pyrethroid-only net Royal Sentry(8.4%).Interestingly,a negative association was found betweenkdrw and the chlorfenapyr-based net Interceptor G2(χ^(2)=138;P<0.0001)with homozygote-resistant mosquitoes predominantly found in the dead ones.Conclusions The high mortality recorded with Interceptor G2 against pyrethroid-resistant malaria vectors in this study provides first semi-field evidence of high efficacy against these major malaria vectors in Cameroon encouraging the implementation of this novel net for malaria control in the country.However,the performance of this net should be established in other locations and on other major malaria vectors before implementation at a large scale.

Comparative study of the effect of solvents on the efficacy of neonicotinoid insecticides against malaria vector populations across Africa

Background:New insecticides with a novel mode of action such as neonicotinoids have recently been recommended for public health by WHO.Resistance monitoring of such novel insecticides requires a robust protocol to monitor the development of resistance in natural populations.In this study,we comparatively used three different solvents to assess the susceptibility of malaria vectors to neonicotinoids across Africa.Methods:Mosquitoes were collected from May to July 2021 from three agricultural settings in Cameroon(Njombe-Penja,Nkolondom,and Mangoum),the Democratic Republic of Congo(Ndjili-Brasserie),Ghana(Obuasi),and Uganda(Mayuge).Using the CDC bottle test,we compared the effect of three different solvents(ethanol,acetone,MERO)on the efficacy of neonicotinoids againstAnopheles gambiae s.l.In addition,TaqMan assays were used to genotype key pyrethroid-resistant markers inAn.gambiae and odds ratio based on Fisher exact test were used to evaluate potential cross-resistance between pyrethroids and clothianidin.Results:Lower mortality was observed when using absolute ethanol or acetone alone as solvent for clothianidin(11.4-51.9% mortality in Nkolondom,31.7-48.2% in Mangoum,34.6-56.1% in Mayuge,39.4-45.6% in Obuasi,83.7-89.3% in Congo and 71.1-95.9% in Njombe pendja)compared to acetone+MERO for which 100% mortality were observed for all the populations.Similar observations were done for imidacloprid and acetamiprid.Synergist assays(PBO,DEM and DEF)with clothianidin revealed a significant increase of mortality suggesting that metabolic resistance mechanisms are contributing to the reduced susceptibility.A negative association was observed between the L1014F-kdr mutation and clothianidin resistance with a greater frequency of homozygote resistant mosquitoes among the dead than among survivors(OR=0.5;P=0.02).However,the I114T-GSTe2 was in contrast significantly associated with a greater ability to survive clothianidin with a higher frequency of homozygote resistant among survivors than other genotypes(OR=2.10;P=0.013).Conclusions:This study revealed a contrasted susceptibility pattern depending on the solvents with ethanol/acetone resulting to lower mortality,thus possibly overestimating resistance,whereas the MERO consistently showed a greater efficacy of neonicotinoids but it could prevent to detect early resistance development.Therefore,we recommend monitoring the susceptibility using both acetone alone and acetone+MERO(4μg/ml for clothianidin)to capture the accurate resistance profile of the mosquito populations.

High insecticide resistance in the major malaria vector Anopheles coluzzii in Chad Republic

Background:The Sahel region of Chad Republic is a prime candidate for malaria pre-elimination.To facilitate preelimination efforts in this region,two populations o f A nopheles coluzzii from Central Chad Republic were characterized,their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.Methods:Bloodfed female Anopheles gom biae s.l.resting indoor,were collected at N'djamena and Massakory,Chad in 2018 and characterized for species composition,and infection rate was determined using the TaqMan assay.Susceptibility to various insecticides was assessed using WHO tube bioassays.Cone bioassays were conducted using various long-lasting insecticidal nets(LLINs).Results were analysed using Chi Square test.Knockdown resistance(kdr)and ace-1 markers were investigated by TaqMan genotyping.Results:Anopheles coluzzi was the major vector found in N’djamena(100%)and Massakory(〜94%).N〇Plasm odium was found in 147 bloodfed F0 An.coluzzii(82 from N'djamena and 65 from Massakory).High intensity pyrethroid resistance was observed with mortalities of<2%for permethrin,deltamethrin and etofenprox,and with<50%and<60%dead following exposure to 10x diagnostic doses of deltamethrin and permethrin,respectively.For both sites,<10%mortalities were observed with DDT.Synergist bioassays with piperonylbutoxide significantly recovered pyrethroid susceptibility in Massakory populations,implicating CYP450s(mortality=13.6%for permethrin,X^2=22.8,df=1,P=0.0006;mortality=13.0%for deltamethrin,x2:8.8,df=1,P<0.00031).Cone-bioassays established complete loss of efficacy of the pyrethroid-based LLINs;and a 100%recovery of susceptibility following exposure to the roof of PermaNet®3.0/containing piperonylbutoxide.Both populations were susceptible to malathion,but high bendiocarb resistance was observed in Massakory population.The absence o f a ce-1 mutation points to the role of metabolic resistance in the bendiocarb resistance.Both 1014F and 1014S mutations were found in both populations at around 60%and<20%respectively.Sequencing of intron-1 of the voltage-gated sodium channel revealed a low genetic diversity suggesting reduced polymorphism.Conclusions:Multiple resistance in An.coluzzii populations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying(IRS)in the Sahel of this country.The pyrethroid-synergists LLINs(e.g.PermaNet®3.0)and organophosphate-based IRS maybe the alternatives for malaria control in this region.

First detection of F1534C knockdown resistance mutation in Aedes aegypti(Diptera:Culicidae)from Cameroon

Background: Aedes borne viral diseases,notably dengue,are increasingly reported in Cameroon with Aedes aegypti being a major vector.Data on insecticide resistance of this vector and underlying mechanisms needed for outbreak preparedness remain scarce in Cameroon.Here,we present the nationwide distribution of insecticide resistance in Ae.aegypti and investigate the potential resistance mechanisms involved.Methods:: Immature stages of Ae.aegypti were collected between March and July 2017 in 13 locations across Cameroon and reared until G1/G2/G3 generation.Larval,adult bioassays,and piperonyl butoxide(PBO)synergist assays were carried out according to World Health Organization guidelines.F1534C mutation was genotyped using allele specific polymerase chain reaction in field collected adults(Go)and the polymorphism of the sodium channel gene was assessed.Theχ2 test was used to compare the mortality rate between bioassays with insecticides only and bioassays after preexposure to PBO synergist.Results: Larval bioassay revealed that all the three populations tested with temephos were susceptible.Adult bioassays showed a good level of susceptibility toward both pyrethroids tested,0.25%permethrin and 0.05%deltamethrin,with six out of 10 populations susceptible.However,two populations(Douala and Edéa)were resistant(deltamethrin[73.2–92.5%mortality],permethrin[2.6–76.3%mortality]).The resistance to 4%dichlorodiphenyltrichloroethane was observed in four out of 10 populations tested(16.8–87.1%mortality).Resistance was also reported to carbamates including 0.1%propoxur(60.8–87.1%mortality)and to 0.1%bendiocarb(82.9%mortality).All populations tested were fully susceptible to 1%fenitrothion.A partial recovery of susceptibility was observed in the pyrethroid resistant population of Douala after pre-exposed to PBO suggesting the implication of cytochrome P450 monoxygenases permethrin resistance.Genotyping and sequencing detected the F1534C kdr mutation in the two pyrethroid resistant locations of Edéa and Douala,with allelic frequency of 3.3%and 33.3%respectively.However,the high genetic diversity of the sodium channel gene supports the recent introduction of this mutation in Cameroon.Conclusions: This study revealed the contrasting resistance profiles to insecticides of Ae.aegypti populations in Cameroon suggesting that,instead of a unique nationwide control approach,a regionally adapted strategy will be needed to control this vector.The localised distribution of the F1534C kdr mutation supports this region-specific control strategy.

Contrasting resistance patterns to type Ⅰ and Ⅱ pyrethroids in two major arbovirus vectors Aedes aegypti and Aedes albopictus in the Republic of the Congo, Central Africa

Background:In the Republic of Congo,with two massive outbreaks of chikungunya observed this decade,little is known about the insecticide resistance profile of the two major arbovirus vectors Aedes aegypti and Aedes albopictus.Here,we established the resistance profile of both species to insecticides and explored the resistance mechanisms to help Congo to better prepare for future outbreaks.Methods:Immature stages o f Ae.aegypti and Ae.albopictus were sampled in May 2017 in eight cities of the Republic of the Congo and reared to adult stage.Larval and adult bioassays,and synergist(piperonyl butoxide[PBO])assays were carried out according to WHO guidelines.F1534C mutation was genotyped in field collected adults in both species and the polymorphism of the sodium channel gene assessed in Ae.oegypti.Results:All tested populations were susceptible to temephos after larval bioassays.A high resistance level was observed to 4%DDT in both species countrywide(21.9-88.3% mortality).All but one population(Ae.oegypti from Ngo)exhibited resistance to type I pyrethroid,permethrin,but showed a full susceptibility to type II pyrethroid(deltamethrin)in almost all locations.Resistance was also reported to 1% propoxur in Ae.aegypti likewise in two Ae.albopictus populations(Owando and Ouesso),and the remaining were fully susceptible.All populations of both species were fully susceptible to 1%fenitrothion.A full recovery of susceptibility was observed in Ae.oegypti and Ae.albopictus when pre-exposed to PBO and then to propoxur and permethrin respectively.The F1534C kdr mutation was not detected in either species.The high genetic variability of the portion of sodium channel spanning the F1534C in Ae.aegypti further supported that knockdown resistance probably play no role in the permethrin resistance.Conclusions:Our study showed that both Aedes species were susceptible to organophosphates(temephos and fenitrothion),while for other insecticide classes tested the profile of resistance vary according to the population origin.These findings could help to implement better and efficient strategies to control these species in the Congo in the advent of future arbovirus outbreaks.