Insecticide Resistance in Malaria Vectors: An Update at a Global Scale

Malaria remains the deadliest vector-borne disease in the world. With nearly half of the world’s population at risk, 216 million people suffered from malaria in 2016, with over 400,000 deaths, mainly in sub-Saharan Africa. Important global efforts have been made to eliminate malaria leading to significant reduction in malaria cases and mortality in Africa by 42% and 66%, respectively. Early diagnosis, improved drug therapies and better health infrastructure are key components, but this extraordinary success is mainly due the use of long-lasting insecticidal nets (LLINs) and indoor residual sprayings (IRS) of insecticide. Unfortunately, the emergence and spread of resistance in mosquito populations against insecticides is jeopardising the effectiveness of the most efficient malaria control interventions. To help establish suitable resistance management strategies, it is vital to better understand the distribution of resistance, its mechanisms and impact on effectiveness of control interventions and malaria transmission. In this chapter, we present the current status of insecticide resistance worldwide in main malaria vectors as well as its impact on malaria transmission, and discuss the molecular mechanisms and future perspectives

Multiple Insecticide Resistance in the Malaria Vector Anopheles funestus from Northern Cameroon Is Mediated by Metabolic Resistance Alongside Potential Target Site Insensitivity Mutations

Background Despite the recent progress in establishing the patterns of insecticide resistance in the major malaria vector Anopheles funestus, Central African populations of this species remain largely uncharacterised. To bridge this important gap and facilitate the implementation of suitable control strategies against this vector, we characterised the resistance patterns of An. funestus population from northern Cameroon. Methods and Findings Collection of indoor-resting female mosquitoes in Gounougou (northern Cameroon) in 2012 and 2015 revealed a predominance of An. funestus during dry season. WHO bioassays performed using F1 An. funestus revealed that the population was multiple resistant to several insecticide classes including pyrethroids (permethrin, deltamethrin, lambda-cyhalothrin and etofenprox), carbamates (bendiocarb) and organochlorines (DDT and dieldrin). However, a full susceptibility was observed against the organophosphate malathion. Bioassays performed with 2015 collection revealed that resistance against pyrethroids and DDT is increasing. PBO synergist assays revealed a significant recovery of susceptibility for all pyrethroids but less for DDT. Analysis of the polymorphism of a portion of the voltage-gated sodium channel gene (VGSC) revealed the absence of the L1014F/S kdr mutation but identified 3 novel amino acid changes I877L, V881L and A1007S. However, no association was established between VGSC polymorphism and pyrethroid/DDT resistance. The DDT resistant 119F-GSTe2 allele (52%) and the dieldrin resistant 296S-RDL allele (45%) were detected in Gounougou. Temporal analysis between 2006, 2012 and 2015 collections revealed that the 119F-GSTe2 allele was relatively stable whereas a significant decrease is observed for 296S-RDL allele. Conclusion This multiple resistance coupled with the temporal increased in resistance intensity highlights the need to take urgent measures to prolong the efficacy of current insecticide-based interventions against An. funestus in this African region

Rapid evolution of pyrethroid resistance prevalence in Anopheles gambiae populations from the cities of Douala and Yaoundé (Cameroon)

Background The adaptation of malaria vectors to urban areas is becoming a serious challenge for malaria control. The study presents the evolution of pyrethroid resistance in mosquito populations from the cities of Douala and Yaoundé between 2010 and 2013. Methods Susceptibility tests to permethrin and deltamethrin were carried out with two- to four-day old unfed Anopheles gambiae sensu lato adults raised from larvae collected from the field. Mosquitoes resistant to permethrin and deltamethrin and control were screened to detect the presence of the kdr alleles using the TaqMan assays. Mosquitoes belonging to the An. gambiae complex were subjected to PCR assays designed for species and molecular forms identifications. The genomic region containing the upstream of intron-1 of the voltage-gated sodium channel was sequenced and compared between mosquitoes originating from different breeding habitats. Results Anopheles gambiae s.l. specimens collected from the city of Douala were all Anopheles coluzzii. In Yaoundé, both An. gambiae and An. coluzzii were recorded. A rapid decrease of mosquito mortality to permethrin and deltamethrin was recorded between 2010 and 2013 in the two cities. The mortality rate varied from 80.3 to 22.3% and 94.4 to 59.7% for permethrin and deltamethrin, respectively. Both kdr alleles L1014F and L1014S were recorded. The frequency of kdr alleles increased rapidly over the study period, varying from 44 to 88.9% in Yaoundé and from 68 to 81% in Douala. The sequencing of a 1,228 bp region of intro-1 of the voltage-gated sodium channel revealed the presence of five different haplotypes. A high number of these haplotypes were recorded in An. coluzzii samples. No evidence for a recent selective sweep on intron-1 sequence within samples originating from different breeding habitat was detected using Fu’s and Tajima Fs statistics. Conclusion The present study supports rapid evolution of pyrethroid resistance in vector populations from the cities of Douala and Yaoundé and calls for immediate action to fight against the increasing prevalence of pyrethroid-resistant mosquitoes

Detection of a reduced susceptibility to chlorfenapyr in the malaria vector Anopheles gambiae contrasts with full susceptibility in Anopheles funestus across Africa

New insecticides have recently been produced to help control pyrethroid-resistant malaria vectors including the pyrrole, chlorfenapyr. Monitoring the susceptibility of mosquito populations against this new product and potential cross-resistance with current insecticides is vital for better resistance management. In this study, we assessed the resistance status of the major malaria vectors Anopheles gambiae and Anopheles funestus to chlorfenapyr across Africa and explored potential cross-resistance with known pyrethroid resistance markers. Efficacy of chlorfenapyr 100 µg/ml against An. gambiae and An. funestus from five Cameroonian locations, the Democratic Republic of Congo, Ghana, Uganda, and Malawi was assessed using CDC bottle assays. Synergist assays were performed with PBO (4%), DEM (8%) and DEF (0.25%) and several pyrethroid-resistant markers were genotyped in both species to assess potential cross-resistance between pyrethroids and chlorfenapyr. Resistance to chlorfenapyr was detected in An. gambiae populations from DRC (Kinshasa) (mortality rate: 64.3 ± 7.1%) Ghana (Obuasi) (65.9 ± 7.4%), Cameroon (Mangoum; 75.2 ± 7.7% and Nkolondom; 86.1 ± 7.4). In contrast, all An. funestus populations were fully susceptible. A negative association was observed between the L1014F-kdr mutation and chlorfenapyr resistance with a greater frequency of homozygote resistant mosquitoes among the dead mosquitoes after exposure compared to alive (OR 0.5; P = 0.02) whereas no association was found between GSTe2 (I114T in An. gambiae; L119F in An. funestus) and resistance to chlorfenapyr. A significant increase of mortality to chlorfenapyr 10 µg/ml was observed in An. funestus after to PBO, DEM and DEF whereas a trend for a decreased mortality was observed in An. gambiae after PBO pre-exposure. This study reveals a greater risk of chlorfenapyr resistance in An. gambiae populations than in An. funestus. However, the higher susceptibility in kdr-resistant mosquitoes points to higher efficacy of chlorfenapyr against the widespread kdr-based pyrethroid resistance

Marked aggravation of pyrethroid resistance in major malaria vectors in Malawi between 2014 and 2021 is partly linked with increased expression of P450 alleles

Background: Increased intensity of pyrethroid resistance is threatening the effectiveness of insecticide-based interventions to control malaria in Africa. Assessing the extent of this aggravation and its impact on the efficacy of these tools is vital to ensure the continued control of major vectors. Here we took advantage of 2009 and 2014 data from Malawi to establish the extent of the resistance escalation in 2021 and assessed its impact on various bed nets performance. Methods: Indoor blood-fed and wild female Anopheles (An) mosquitoes were collected with an electric aspirator in Chikwawa. Cocktail and SINE PCR were used to identify sibling species belonging to An. funestus group and An. gambiae complex. The susceptibility profile to the four classes of insecticides was assessed using the WHO tubes bioassays. Data were saved in an Excel file. Analysis was done using Vassarstats and figures by Graph Pad. Results: In this study, a high level of resistance was observed with pyrethroids (permethrin, deltamethrin and alpha-cypermethrin with mortality rate at 5x discriminating concentration (DC) < 50% and Mortality rate at 10x DC < 70%). A high level of resistance was also observed to carbamate (bendiocarb) with mortality rate at 5x DC < 25%). Aggravation of resistance was also noticed between 2009 and 2021. For pyrethroids, the mortality rate for permethrin reduced from 47.2% in 2009 to 13% in 2014 and 6.7% in 2021. For deltamethrin, the mortality rate reduced from 42.3% in 2009 to 1.75% in 2014 and 5.2% in 2021. For Bendiocarb, the mortality rate reduced from 60% in 2009 to 30.1% in 2014 and 12.2% in 2021. The high resistance observed is consistent with a drastic loss of pyrethroid-only bed nets efficacy although Piperonyl butoxide (PBO)-based nets remain effective. The resistance pattern observed was linked with high up-regulation of the P450 genes CYP6P9a, CYP6P9b and CYP6M7 in An. funestus s.s. mosquitoes surviving exposure to deltamethrin at 1x, 5x and 10x DC. A significant association was observed between the 6.5 kb structural variant and resistance escalation with homozygote resistant (SV+/SV+) more likely to survive exposure to 5x and 10x (OR = 4.1; P < 0.001) deltamethrin than heterozygotes. However, a significant proportion of mosquitoes survived the synergist assays with PBO suggesting that other mechanisms than P450s are present. Conclusions: This resistance aggravation in An. funestus s.s. Malawian population highlights an urgent need to deploy novel control tools not relying on pyrethroids to improve the effectiveness of vector control

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 on Anopheles gambiae s.l. and Anopheles 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-resistant An. 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-seeking An. 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 between kdrw and the chlorfenapyr-based net Interceptor G2 (χ2 = 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

Entomological longitudinal surveys in two contrasted eco-climatic settings in Cameroon reveal a high malaria transmission from Anopheles funestus associated with GSTe2 metabolic resistance

Background: The impact of metabolic resistance to insecticides on malaria transmission remains poorly characterised notably through application of entomological parameters. The lack of resistance markers has been one of the limiting factors preventing a robust assessment of such impact. To this end, the present study sought to investigate how the L119F-Gste2 metabolic gene influences entomological parameters underpinning mosquitos’ propensity to transmit Plasmodium spp. Methods: Longitudinal studies were carried out in Mibellon and Elende, two different eco-climatic settings in Cameroon and mosquitoes were collected using Human Landing Catch (HLC), Centre for Disease Control Light Trap (CDC-LT) and Pyrethrum Spray Catch (PSC) technics. Plasmodium sporozoite parasites were detected by TaqMan and Nested PCR, and blood meal origin by ELISA. The allele-specific PCR (AS-PCR) method was used to genotype the L119F-GSTe2 marker and association with malaria transmission was established by comparing key transmission parameters such as the Entomological Inoculation Rate (EIR) between individuals with different L119F-GSTe2 genotypes. Results: An. funestus s.l was the predominant malaria vector collected during the entomological survey in both sites (86.6% and 96.4% in Elende and Mibellon, respectively) followed by An. gambiae s.l (7.5% and 2.4%, respectively). Sporozoite infection rates were very high in both collection sites (8.7% and 11% in Elende and Mibellon, respectively). An. funestus s.s exhibited a very high entomological inoculation rate (EIR) (66 ib/h/month and 792 ib/h/year) and was responsible for 98.6% of all malaria transmission events occurring in both sites. The Human Blood Index was also high in both locations (HBI = 94%). An. funestus s.s. mosquitoes with both 119 F/F (RR) and L119F (RS) genotypes had a significantly higher transmission intensity than their susceptible L/L119 (SS) counterparts (IRR = 2.2, 95%CI (1.1–5.2), p = 0.03; IRR = 2.5, 95% CI (1.2–5.8), p = 0.01 respectively). Conclusion: This study highlights the major role that An. funestus s.s plays in malaria transmission in Cameroon with an aggravation from GSTe2-based metabolic resistance

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 against Anopheles gambiae s.l. In addition, TaqMan assays were used to genotype key pyrethroid-resistant markers in An. 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 (4 µg/ml for clothianidin) to capture the accurate resistance profile of the mosquito populations. Graphical Abstract

Exploring the molecular mechanisms of increased intensity of pyrethroid resistance in Central African population of a major malaria vector Anopheles coluzzii

Molecular mechanisms driving the escalation of pyrethroid resistance in the major malaria mosquitoes of Central Africa remain largely uncharacterized, hindering effective management strategies. Here, resistance intensity and the molecular mechanisms driving it were investigated in a population of Anopheles coluzzii from northern Cameroon. High levels of pyrethroid and organochloride resistance were observed in An. coluzzii population, with no mortality for 1× permethrin; only 11% and 33% mortalities for 5× and 10× permethrin diagnostic concentrations, and <2% mortalities for deltamethrin and DDT, respectively. Moderate bendiocarb resistance (88% mortality) and full susceptibility to malathion were observed. Synergist bioassays with piperonyl butoxide recovered permethrin susceptibility, with mortalities increasing to 53.39%, and 87.30% for 5× and 10× permethrin, respectively, implicating P450 monooxygenases. Synergist bioassays with diethyl maleate (DEM) recovered permethrin and DDT susceptibilities (mortalities increasing to 34.75% and 14.88%, respectively), implicating glutathione S‐transferases. RNA‐seq‐based genome‐wide transcriptional analyses supported by quantitative PCR identified glutathione S‐transferase, GSTe2 (RNA‐seqFC = 2.93 and qRT‐PCRFC = 8.4, p < 0.0043) and CYP450, CYP6Z2 (RNA‐seqFC = 2.39 and qRT‐PCRFC = 11.7, p < 0.0177) as the most overexpressed detoxification genes in the pyrethroid‐resistant mosquitoes, compared to mosquitoes of the susceptible Ngousso colony. Other overexpressed genes include P450s, CYP6M2 (FC = 1.68, p < 0.0114), CYP4G16 (FC = 2.02, p < 0.0005), and CYP4G17 (FC = 1.86, p < 0.0276). While high frequency of the 1014F kdr mutation (50%) and low frequencies of 1014S (6.61%) and 1575Y (10.29%) were observed, no ace‐1 mutation was detected in bendiocarb‐resistant populations, suggesting the preeminent role of metabolic mechanism. Overexpression of metabolic resistance genes (including GSTe2 and CYP6Z2 known to confer resistance to multiple insecticides) in An. coluzzii from the Sudan Savannah of Cameroon highlights the need for alternative management strategies to reduce malaria burden in northern Cameroon

Escalating pyrethroid resistance in two major malaria vectors Anopheles funestus and Anopheles gambiae (s.l.) in Atatam, Southern Ghana

Background: Aggravation of insecticide resistance in malaria vectors is threatening the efforts to control malaria by reducing the efficacy of insecticide-based interventions hence needs to be closely monitored. This study investigated the intensity of insecticide resistance of two major malaria vectors An. funestus sensu stricto (s.s.) and An. gambiae sensu lato (s.l.) collected in southern Ghana and assessed the bio-efficacy of several long-lasting insecticidal nets (LLINs) against these mosquito populations. Methods: The insecticide susceptibility profiles of Anopheles funestus s.s. and Anopheles gambiae s.l. populations from Obuasi region (Atatam), southern Ghana were characterized and the bio-efficacy of some LLINs was assessed to determine the impact of insecticide resistance on the effectiveness of these tools. Furthermore, molecular markers associated with insecticide resistance in both species were characterized in the F0 and F1 populations using PCR and qPCR methods. Results: Anopheles funestus s.s. was the predominant species and was resistant to pyrethroids, organochlorine and carbamate insecticides, but fully susceptible to organophosphates. An. gambiae s.l. was resistant to all four insecticide classes. High intensity of resistance to 5 × and 10 × the discriminating concentration (DC) of pyrethroids was observed in both species inducing a considerable loss of efficacy of long-lasting insecticidal nets (LLINs). Temporal expression analysis revealed a massive 12-fold increase in expression of the CYP6P4a cytochrome P450 gene in An. funestus s.s., initially from a fold change of 41 (2014) to 500 (2021). For both species, the expression of candidate genes did not vary according to discriminating doses. An. gambiae s.l. exhibited high frequencies of target-site resistance including Vgsc-1014F (90%) and Ace-1 (50%) while these mutations were absent in An. funestus s.s. Conclusions: The multiple and high intensity of resistance observed in both malaria vectors highlights the need to implement resistance management strategies and the introduction of new insecticide chemistries