Insecticide Resistance In Malaria Vectors Research Articles

Spatio-temporal characterization of phenotypic resistance in malaria vector species.

Malaria, a deadly disease caused by Plasmodium protozoa parasite and transmitted through bites of infected female Anopheles mosquitoes, remains a significant public health challenge in sub-Saharan Africa. Efforts to eliminate malaria have increasingly focused on vector control using insecticides. However, the emergence of insecticide resistance (IR) in malaria vectors pose a formidable obstacle, and the current IR mapping models remain static, relying on fixed coefficients. This study introduces a dynamic spatio-temporal approach to characterize phenotypic resistance in Anopheles gambiae complex and Anopheles arabiensis. We developed a cellular automata (CA) model and applied it to data collected from Ethiopia, Nigeria, Cameroon, Chad, and Burkina Faso. The data encompasses georeferenced records detailing IR levels in mosquito vector populations across various classes of insecticides. In characterizing the dynamic patterns of confirmed resistance, we identified key driving factors through correlation analysis, chi-square tests, and extensive literature review. The CA model demonstrated robustness in capturing the spatio-temporal dynamics of confirmed IR states in the vector populations. In our model, the key driving factors included insecticide usage, agricultural activities, human population density, Land Use and Land Cover (LULC) characteristics, and environmental variables. The CA model developed offers a robust tool for countries that have limited data on confirmed IR in malaria vectors. The embrace of a dynamical modeling approach and accounting for evolving conditions and influences, contribute to deeper understanding of IR dynamics, and can inform effective strategies for malaria vector control, and prevention in regions facing this critical health challenge.

The impact of agrochemical pollutant mixtures on the selection of insecticide resistance in the malaria vector Anopheles gambiae: insights from experimental evolution and transcriptomics

BackgroundThere are several indications that pesticides used in agriculture contribute to the emergence and spread of resistance of mosquitoes to vector control insecticides. However, the impact of such an indirect selection pressure has rarely been quantified and the molecular mechanisms involved are still poorly characterized. In this context, experimental selection with different agrochemical mixtures was conducted in Anopheles gambiae. The multi-generational impact of agrochemicals on insecticide resistance was evaluated by phenotypic and molecular approaches.MethodsMosquito larvae were selected for 30 generations with three different agrochemical mixtures containing (i) insecticides, (ii) non-insecticides compounds, and (iii) both insecticide and non-insecticide compounds. Every five generations, the resistance of adults to deltamethrin and bendiocarb was monitored using bioassays. The frequencies of the kdr (L995F) and ace1 (G119S) target-site mutations were monitored every 10 generations. RNAseq was performed on all lines at generation 30 in order to identify gene transcription level variations and polymorphisms associated with each selection regime.ResultsLarval selection with agrochemical mixtures did not affect bendiocarb resistance and did not select for ace1 mutation. Contrastingly, an increased deltamethrin resistance was observed in the three selected lines. Such increased resistance was not majorly associated with the presence of kdr L995F mutation in selected lines. RNA-seq identified 63 candidate resistance genes over-transcribed in at least one selected line. These include genes coding for detoxification enzymes or cuticular proteins previously associated with insecticide resistance, and other genes potentially associated with chemical stress response. Combining an allele frequency filtering with a Bayesian FST-based genome scan allowed to identify genes under selection across multiple genomic loci, supporting a multigenic adaptive response to agrochemical mixtures.ConclusionThis study supports the role of agrochemical contaminants as a significant larval selection pressure favouring insecticide resistance in malaria vectors. Such selection pressures likely impact kdr mutations and detoxification enzymes, but also more generalist mechanisms such as cuticle resistance, which could potentially lead to cross-tolerance to unrelated insecticide compounds. Such indirect effect of global landscape pollution on mosquito resistance to public health insecticides deserves further attention since it can affect the nature and dynamics of resistance alleles circulating in malaria vectors and impact the efficacy of control vector strategies.

Large and complete datasets, and modelling reveal the major determinants of physiological and behavioral insecticide resistance of malaria vectors

Large and complete datasets, and modelling reveal the major determinants of physiological and behavioral insecticide resistance of malaria vectors

Genomic surveillance of Anopheles mosquitoes on the Bijagós Archipelago using custom targeted amplicon sequencing identifies mutations associated with insecticide resistance

BackgroundInsecticide resistance is reducing the efficacy of vector control interventions, consequently threatening efforts to control vector-borne diseases, including malaria. Investigating the prevalence of molecular markers of resistance is a useful tool for monitoring the spread of insecticide resistance in disease vectors. The Bijagós Archipelago (Bijagós) in Guinea-Bissau is a region of stable malaria transmission where insecticide-treated nets are the mainstay for malaria control. However, the prevalence of molecular markers of insecticide resistance in malaria vectors is not well understood.MethodsA total of 214 Anopheles mosquitoes were analysed from 13 islands across the Bijagós. These mosquitoes were collected using CDC light traps in November 2019, during the peak malaria transmission season. High-throughput multiplex amplicon sequencing was used to investigate the prevalence of 17 different molecular markers associated with insecticide resistance in four genes: vgsc, rdl, ace1 and gste2.ResultsOf the 17 screened mutations, four were identified in mosquitoes from the Bijagós: vgsc L995F (12.2%), N1570Y (6.2%) and A1746S (0.7%) and rdl A269G (1.1%). This study is the first to report the L995F knock-down resistance (kdr)-west allele in Anopheles melas on the Archipelago. An additional eight non-synonymous single-nucleotide polymorphisms were identified across the four genes which have not been described previously. The prevalences of the vgsc L995F and N1570Y mutations were higher on Bubaque Island than on the other islands in this study; Bubaque is the most populous island in the archipelago, with the greatest population mobility and connection to continental Guinea-Bissau.ConclusionsThis study provides the first surveillance data for genetic markers present in malaria vectors from islands across the Bijagós Archipelago. Overall prevalence of insecticide resistance mutations was found to be low. However, the identification of the vgsc L995F and N1570Y mutations associated with pyrethroid resistance warrants further monitoring. This is particularly important as the mainstay of malaria control on the islands is the use of pyrethroid insecticide-treated nets.Graphical

Differential insecticide resistance in Anopheles arabiensis populations in the seaside area of Mbour and its suburbs in Senegal

Regular monitoring of insecticide resistance status is an important step in implementing appropriate and adapted insecticide-based strategies for vector control. In Senegal, Indoor Residual Spraying (IRS) and a national distribution campaign for long-lasting insecticide-treated net (LLIN) have been implemented since 2007 and 2009, respectively to prevent malaria transmission. To expand and ensure the sustainability of these strategies, we conducted a study on the status of insecticide resistance in malaria vectors in the seaside area of Mbour and its suburbs where no data were previously available.Anopheles larvae were sampled from four study sites (two in both coastal and inland areas) and reared to adulthood in the insectarium. Non-blood-fed females aged 3–5 days were then tested for susceptibility to permethrin, deltamethrin, lambdacyhalothrin, bendiocarb and pirimiphos-methyl. PCR amplification was used to identify sibling species of the An. gambiae complex and genotyping for the presence of resistance knockdown (kdr) L1014S, L1014F and Ace-1 G119S.Anopheles arabiensis was the only species present in the area. At all four sites, mosquitoes were resistant to deltamethrin, permethrin, and lambdacyhalothrin, and exhibited varying degrees of resistance to bendiocarb and pirimiphos-methyl. Overall, high levels of leucine-serine/phenylalanine substitutions at position 1014 (L1014S/L1014F) were observed, with frequencies ranging from 76.4 to 85.2 % for L1014F, and from 43.2 to 66.7 % for L1014S, compared to 8.1 to 28.3 for the Ace-1 G119S mutation.These results indicate a high level of phenotypic and genotypic resistance to insecticides, which is alarming, as it could have a significant impact on the operational effectiveness of current vector control tools that rely on pyrethroids. However, in the case of bendiocarb and pirimiphos-methyl, while some level of tolerance was observed, their potential use requires regular monitoring to prevent operational failure, as their deployment could potentially lead to an increase in resistance to them.

Evidence of intensification of pyrethroid resistance in the major malaria vectors in Kinshasa, Democratic Republic of Congo

Assessing patterns and evolution of insecticide resistance in malaria vectors is a prerequisite to design suitable control strategies. Here, we characterised resistance profile in Anopheles gambiae and Anopheles funestus in Kinshasa and assess the level of aggravation by comparing to previous 2015 estimates. Both species collected in July 2021 were highly resistant to pyrethroids at 1×, 5× and 10× concentrations (mortality < 90%) and remain fully susceptible to bendiocarb and pirimiphos methyl. Compared to 2015, Partial recovery of susceptibility was observed in A. gambiae after PBO synergist assays for both permethrin and α-cypermethrin and total recovery of susceptibility was observed for deltamethrin in 2021. In addition, the efficacy of most bednets decreased significantly in 2021. Genotyping of resistance markers revealed a near fixation of the L1014-Kdr mutation (98.3%) in A. gambiae in 2021. The frequency of the 119F-GSTe2 resistant significantly increased between 2015 and 2021 (19.6% vs 33.3%; P = 0.02) in A. funestus. Transcriptomic analysis also revealed a significant increased expression (P < 0.001) of key cytochrome P450s in A. funestus notably CYP6P9a. The escalation of pyrethroid resistance observed in Anopheles populations from Kinshasa coupled with increased frequency/expression level of resistance genes highlights an urgent need to implement tools to improve malaria vector control.

Effects of next-generation, dual-active-ingredient, long-lasting insecticidal net deployment on insecticide resistance in malaria vectors in Tanzania: an analysis of a 3-year, cluster-randomised controlled trial

Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance. For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616). Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial. Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies. UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium.

Recombinant expression and characterization of GSTd3 from a resistant population of Anopheles arabiensis and comparison of DDTase activity with GSTe2

The development of insecticide resistance in malaria vectors is a challenge for the global effort to control and eradicate malaria. Glutathione S-transferases (GSTs) are multifunctional enzymes involved in the detoxification of many classes of insecticides. For mosquitoes, it is known that overexpression of an epsilon GST, GSTe2, confers resistance towards DDT and pyrethroids. In addition to GSTe2, consistent overexpression of a delta class GST, GSTd3, has been observed in insecticide resistant populations of different malaria vector species. However, the functional role of GSTd3 towards DDT resistance has not yet been investigated. Here, we recombinantly expressed both GSTe2 and GSTd3 from Anopheles arabiensis and compared their metabolic activities against DDT. Both AaGSTd3 and AaGSTe2 exhibited CDNB-conjugating and glutathione peroxidase activity and DDT metabolism was observed for both GSTs. However, the DDT dehydrochlorinase activity exhibited by AaGSTe2 was much higher than for AaGSTd3, and AaGSTe2 was also able to eliminate DDE although the metabolite could not be identified. Molecular modeling revealed subtle differences in the binding pocket of both enzymes and a better fit of DDT within the H-site of AaGSTe2. The overexpression but much lower DDT metabolic activity of AaGSTd3, might suggest that AaGSTd3 sequesters DDT. These findings highlight the complexity of insecticide resistance in the major malaria vectors and the difficulties associated with control of the vectors using DDT, which is still used for indoor residual spraying.

Species composition and insecticide resistance in malaria vectors in Ellibou, southern Côte d’Ivoire and first finding of Anopheles arabiensis in Côte d’Ivoire

BackgroundKnowing the species composition and insecticide resistance status of the target vector population is important to guide malaria vector control. The aim of this study was to characterize the malaria vector population in terms of species composition, insecticide susceptibility status and potential underlying resistance mechanisms in Ellibou, southern Côte d’Ivoire.MethodsA 1-year longitudinal entomological survey was conducted using light traps and pyrethroid spray catches to sample adult mosquitoes in combination with larval sampling. The susceptibility status of Anopheles gambiae sensu lato (s.l.) to bendiocarb, deltamethrin, DDT and malathion was assessed using the World Health Organization insecticide susceptibility test. Additionally, An. gambiae specimens were screened for knockdown (kdr) and acetylcholineesterase (ace1) target site resistance alleles, and the expression levels of eight metabolic resistance genes, including seven cytochrome P450 monooxygenases (P450s) and one glutathione S-transferase (GST), measured with reverse transcription quantitative real-time polymerase chain reaction (qPCR).ResultsOverall, 2383 adult mosquitoes from 12 different taxa were collected with Culex quinquefasciatus and An. gambiae being the predominant taxa. Molecular identification of An. gambiae s.l. revealed the presence of Anopheles arabiensis, Anopheles coluzzii, An. gambiae sensu stricto (s.s.) and Anopheles coluzzii/An. gambiae s.s. hybrids. Anopheles gambiae mosquitoes were resistant to all insecticides except malathion. PCR diagnostics revealed the presence of ace1-G280S and the kdr L995F, L995S and N1570Y target-site mutations. Additionally, several genes were upregulated, including five P450s (i.e., CYP6P3, CYP6M2, CYP9K1, CYP6Z1, CYP6P1) and GSTE2.ConclusionThis is the first documented presence of An. arabiensis in Côte d’Ivoire. Its detection – together with a recent finding further north of the country – confirms its existence in the country, which is an early warning sign, as An. arabiensis shows a different biology than the currently documented malaria vectors. Because the local An. gambiae population was still susceptible to malathion, upregulation of P450s, conferring insecticide resistance to pyrethroids, together with the presence of ace1, suggest negative cross-resistance. Therefore, organophosphates could be an alternative insecticide class for indoor residual spraying in the Ellibou area, while additional tools against the outdoor biting An. arabiensis will have to be considered.

Contrasting Patterns of Asaia Association with Pyrethroid Resistance Escalation between the Malaria Vectors Anopheles funestus and Anopheles gambiae

Microbiome composition has been associated with insecticide resistance in malaria vectors. However, the contribution of major symbionts to the increasingly reported resistance escalation remains unclear. This study explores the possible association of a specific endosymbiont, Asaia spp., with elevated levels of pyrethroid resistance driven by cytochrome P450s enzymes and voltage-gated sodium channel mutations in Anopheles funestus and Anopheles gambiae. Molecular assays were used to detect the symbiont and resistance markers (CYP6P9a/b, 6.5 kb, L1014F, and N1575Y). Overall, genotyping of key mutations revealed an association with the resistance phenotype. The prevalence of Asaia spp. in the FUMOZ_X_FANG strain was associated with the resistance phenotype at a 5X dose of deltamethrin (OR = 25.7; p = 0.002). Mosquitoes with the resistant allele for the markers tested were significantly more infected with Asaia compared to those possessing the susceptible allele. Furthermore, the abundance correlated with the resistance phenotype at 1X concentration of deltamethrin (p = 0.02, Mann-Whitney test). However, for the MANGOUM_X_KISUMU strain, findings rather revealed an association between Asaia load and the susceptible phenotype (p = 0.04, Mann-Whitney test), demonstrating a negative link between the symbiont and permethrin resistance. These bacteria should be further investigated to establish its interactions with other resistance mechanisms and cross-resistance with other insecticide classes.

Entomological monitoring data driving decision-making for appropriate and sustainable malaria vector control in Côte d’Ivoire

BackgroundEntomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d’Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations.To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity.ResultsPirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites.Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n).ConclusionBased on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

The Effect of Plasmodium falciparum (Welch) (Haemospororida: Plasmodiidae) Infection on the Susceptibility of Anopheles gambiae s.l. and Anopheles funestus (Diptera: Culicidae) to Pyrethroid Insecticides in the North-Western and South-Eastern, Tanzania.

The rapid development of insecticide resistance in malaria vectors threatens insecticide-based interventions. It is hypothesized that infection of insecticide-resistant vectors with Plasmodium parasites increases their vulnerability to insecticides, thus assuring the effectiveness of insecticide-based strategies for malaria control. Nonetheless, there is limited field data to support this. We investigated the effect of the Plasmodium falciparum infection on the susceptibility of Anopheles gambiae s.l. and Anopheles funestus to pyrethroids in south-eastern (Kilombero) and north-western (Muleba), Tanzania. The wild-collected mosquitoes were tested against 0.05% deltamethrin and 0.75% permethrin, then assessed for sporozoite rate and resistant gene (kdr) mutations. All Anopheles gambiae s.l. from Kilombero were An. arabiensis (Patton, 1905) while those from Muleba were 87% An. gambiae s.s (Giles, 1902) and 13% An. Arabiensis. High levels of pyrethroid resistance were observed in both areas studied. The kdr mutation was only detected in An. gambiae s.s. at the frequency of 100% in survivors and 97% in dead mosquitoes. The P. falciparum sporozoite rates were slightly higher in susceptible than in resistant mosquitoes. In Muleba, sporozoite rates in An. gambiae s.l. were 8.1% and 6.4% in dead mosquitoes and survivors, respectively (SRR = 1.28, p = 0.19). The sporozoite rates in Kilombero were 1.3% and 0.7% in the dead and survived mosquitoes, respectively (sporozoite rate ratio (SRR) = 1.9, p = 0.33). In An. funestus group sporozoite rates were 6.2% and 4.4% in dead and survived mosquitoes, respectively (SRR = 1.4, p = 0.54). These findings indicate that insecticides might still be effective in malaria control despite the rapid development of insecticide resistance in malaria vectors.

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

BackgroundAggravation 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.MethodsThe 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.ResultsAnopheles 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.ConclusionsThe 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.

Multidisciplinary Investigations of Sustained Malaria Transmission in the Greater Mekong Subregion.

In the course of malaria elimination in the Greater Mekong Subregion (GMS), malaria epidemiology has experienced drastic spatiotemporal changes with residual transmission concentrated along international borders and the rising predominance of Plasmodium vivax. The emergence of Plasmodium falciparum parasites resistant to artemisinin and partner drugs renders artemisinin-based combination therapies less effective while the potential spread of multidrug-resistant parasites elicits concern. Vector behavioral changes and insecticide resistance have reduced the effectiveness of core vector control measures. In recognition of these problems, the Southeast Asian International Center of Excellence for Malaria Research (ICEMR) has been conducting multidisciplinary research to determine how human migration, antimalarial drug resistance, vector behavior, and insecticide resistance sustain malaria transmission at international borders. These efforts allow us to comprehensively understand the ecology of border malaria transmission and develop population genomics tools to identify and track parasite introduction. In addition to employing invivo, invitro, and molecular approaches to monitor the emergence and spread of drug-resistant parasites, we also use genomic and genetic methods to reveal novel mechanisms of antimalarial drug resistance of parasites. We also use omics and population genetics approaches to study insecticide resistance in malaria vectors and identify changes in mosquito community structure, vectorial potential, and seasonal dynamics. Collectively, the scientific findings from the ICEMR research activities offer a systematic view of the factors sustaining residual malaria transmission and identify potential solutions to these problems to accelerate malaria elimination in the GMS.

Participatory approaches for raising awareness among subsistence farmers in Tanzania about the spread of insecticide resistance in malaria vectors and the possible link to improper agricultural pesticide use

BackgroundInsecticide resistance is a key barrier to long-term malaria control, and it may be exacerbated by poor agricultural pesticide use. Current practices, however, do not link public health and agricultural pesticide use. This study investigated the perspectives of farmers and other stakeholders regarding the integration of agricultural and public health measures to address resistance. Additionally, the feasibility of participatory workshops to increase the farmers’ understanding and participation in pesticide stewardship was assessed.MethodsFour themes were investigated: pesticide awareness, practices, and opinions of; insecticide resistance in malaria vectors; the effectiveness of current malaria prevention tools; and the links between agricultural and public health pesticide usage. Participatory workshops and field training were held with entomologists, farmers, and agricultural specialists, focusing on agro-ecosystem practices related to pest control; and local farmers were involved in live-testing for insecticides resistance of local Anopheles mosquitoes.ResultsMost farmers (94%) considered pesticides effective, and nearly half of them (n = 198, 46.4%) could identify and name crop pests and diseases, mostly using local names. Three quarters were unaware of mosquito larvae in their fields, and only 7% considered their fields as potential sources of mosquitoes. Two thirds were uninformed of any effects that agricultural pesticides may have on mosquitoes, and three quarters had never heard of resistance in malaria mosquitoes. Experts from various sectors acknowledged that agricultural pesticides might impact malaria control through increasing resistance. They did, however, emphasize the importance of crop protection and advocated for the use of pesticides sparingly and non-chemical approaches. Farmers learnt how to discriminate between malaria vectors and non-vectors, identify agricultural pests and diseases, choose and use pesticides effectively, and conduct resistance tests during the participatory workshops.ConclusionThis study emphasizes the significance of enhancing subsistence farmers’ awareness of mosquito ecology as well as merging public health and agricultural pest management measures. Participatory techniques have the potential to raise stakeholder awareness and engagement, resulting in more effective resistance management.

Susceptibility status of the malaria vector, Anopheles arabiensis to insecticides used in vector-borne diseases control in areas with heterogeneous sources of pollutants in South-East Tanzania

In malaria-endemic countries, monitoring of insecticide resistance in malaria vectors in areas contaminated with different sources of pollutants is still lacking. This study investigated insecticide resistance and its mechanisms in Anopheles arabiensis across four villages in Southeast Tanzania. Mosquitoes were collected from breeding habitats and tested against permethrin, deltamethrin, lambda-cyhalothrin, pirimiphos-methyl, and bendiocarb. Mechanisms of resistance [P450s enzymes and knockdown resistance (kdr)] were investigated. Comparison of mosquito mortality between sites was performed through one-way analysis of variance (ANOVA) and Tukey–Kramer tests. Anopheles arabiensis was highly resistant to pyrethroid and fully susceptible to bendiocarb and pirimiphos-methyl with mortality ranges of 29%−57%; 37%−65%; 34%−53%; 98%−100%; and 100% in permethrin, deltamethrin, lambda-cyhalothrin, pirimiphos-methyl, and bendiocarb respectively. Anopheles arabiensis mortality, particularly, in permethrin and deltamethrin was significantly higher in less polluted areas (Mofu and Namwawala) p < 0.001 as compared to areas with agrochemicals (Minepa) and domestic pollutants (Ifakara town) (p < 0.01). Moreover, significant involvement of P450s enzymes in pyrethroid resistance was observed while kdr mutations were not detected. The present study shows that pollution of mosquito breeding habitats might accelerate insecticide resistance which could impede the control of malaria vectors.

The resting behavior of malaria vectors in different ecological zones of Ghana and its implications for vector control

BackgroundIn sub-Saharan Africa there is widespread use of long-lasting insecticidal nets and indoor residual spraying to help control the densities of malaria vectors and decrease the incidence of malaria. This study was carried out to investigate the resting behavior, host preference and infection with Plasmodium falciparum of malaria vectors in Ghana in the context of the increasing insecticide resistance of malaria vectors in sub-Saharan Africa.MethodsIndoor and outdoor resting anopheline mosquitoes were sampled during the dry and rainy seasons in five sites in three ecological zones [Sahel savannah (Kpalsogo, Pagaza, Libga); coastal savannah (Anyakpor); and forest (Konongo)]. Polymerase chain reaction-based molecular diagnostics were used to determine speciation, genotypes for knockdown resistance mutations (L1014S and L1014F) and the G119S ace1 mutation, specific host blood meal origins and sporozoite infection in the field-collected mosquitoes.ResultsAnopheles gambiae sensu lato (s.l.) predominated (89.95%, n = 1718), followed by Anopheles rufipes (8.48%, n = 162) and Anopheles funestus s.l. (1.57%, n = 30). Sibling species of the Anopheles gambiae s.l. revealed Anopheles coluzzii accounted for 63% (95% confidence interval = 57.10–68.91) and 27% (95% confidence interval = 21.66–32.55) was Anopheles gambiae s. s.. The mean resting density of An. gambiae s.l. was higher outdoors (79.63%; 1368/1718) than indoors (20.37%; 350/1718) (Wilcoxon rank sum test, Z = − 4.815, P < 0.0001). The kdr west L1014F and the ace1 mutation frequencies were higher in indoor resting An. coluzzii and An. gambiae in the Sahel savannah sites than in the forest and coastal savannah sites. Overall, the blood meal analyses revealed that a larger proportion of the malaria vectors preferred feeding on humans (70.2%) than on animals (29.8%) in all of the sites. Sporozoites were only detected in indoor resting An. coluzzii from the Sahel savannah (5.0%) and forest (2.5%) zones.ConclusionsThis study reports high outdoor resting densities of An. gambiae and An. coluzzii with high kdr west mutation frequencies, and the presence of malaria vectors indoors despite the use of long-lasting insecticidal nets and indoor residual spraying. Continuous monitoring of changes in the resting behavior of mosquitoes and the implementation of complementary malaria control interventions that target outdoor resting Anopheles mosquitoes are necessary in Ghana.Graphical abstract

Rapid evolution of insecticide resistance and patterns of pesticides usage in agriculture in the city of Yaoundé, Cameroon

BackgroundThe practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon.MethodsWHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage.ResultsHigh resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to < 23% in 2019 for deltamethrin and permethrin and from 97% in 2017 to < 86% in 2019 for bendiocarb). Both An. gambiae (s.s.) and An. coluzzii were recorded. Over 150 pesticides and fertilizers were sold by retailers for agricultural purposes in the city of Yaoundé. Most farmers do not respect safety practices. Poor practices including extensive and inappropriate application of pesticides as well as poor management of perished pesticides and empty pesticide containers were also documented.ConclusionsThe study indicated rapid evolution of insecticide resistance and uncontrolled usage of pesticides by farmers in agriculture. There is an urgent need to address these gaps to improve the management of insecticide resistance.Graphical

Community structure and insecticide resistance of malaria vectors in northern-central Myanmar

BackgroundMyanmar is one of the six countries in the Greater Mekong Subregion (GMS) of Southeast Asia. Malaria vectors comprise many Anopheles species, which vary in abundance and importance in malaria transmission among different geographical locations in the GMS. Information about the species composition, abundance, and insecticide resistance status of vectorial systems in Myanmar is scarce, hindering our efforts to effectively control malaria vectors in this region.MethodsDuring October and November 2019, larvae and adult females of Anopheles mosquitoes were collected in three sentinel villages of Banmauk township in northern Myanmar. Adult female mosquitoes collected by cow-baited tent collection (CBTC) and adults reared from field-collected larvae (RFCL) were used to determine mortality rates and knockdown resistance (kdr) against deltamethrin using the standard WHO susceptibility test. Molecular species identification was performed by multiplex PCR and ITS2 PCR, followed by DNA sequencing. The kdr mutation at position 1014 of the voltage-gated sodium channel gene was genotyped by DNA sequencing for all Anopheles species tested.ResultsA total of 1596 Anopheles mosquitoes from seven morphologically identified species groups were bioassayed. Confirmed resistance to deltamethrin was detected in the populations of An. barbirostris (s.l.), An. hyrcanus (s.l.), and An. vagus, while possible resistance was detected in An. annularis (s.l.), An. minimus, and An. tessellatus. Anopheles kochi was found susceptible to deltamethrin. Compared to adults collected by CBTC, female adults from RFCL had significantly lower mortality rates in the four species complexes. A total of 1638 individuals from 22 Anopheles species were molecularly identified, with the four most common species being An. dissidens (20.5%) of the Barbirostris group, An. peditaeniatus (19.4%) of the Hyrcanus group, An. aconitus (13.4%) of the Funestus group, and An. nivipes (11.5%) of the Annularis group. The kdr mutation L1014F was only detected in the homozygous state in two An. subpictus (s.l.) specimens and in a heterozygous state in one An. culicifacies (s.l.) specimen.ConclusionsThis study provides updated information about malaria vector species composition and insecticide resistance status in northern Myanmar. The confirmed deltamethrin resistance in multiple species groups constitutes a significant threat to malaria vector control. The lack or low frequency of target-site resistance mutations suggests that other mechanisms are involved in resistance. Continual monitoring of the insecticide resistance of malaria vectors is required for effective vector control and insecticide resistance management.Graphical

A new generation of long-lasting insecticidal nets

The scale-up of malaria interventions over the past two decades has resulted in substantial reductions in malaria burden, especially in sub-Saharan Africa, which accounts for 95% of the global malaria burden. 1 Bhatt S Weiss DJ Cameron E et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015; 526: 207-211 Crossref PubMed Scopus (1439) Google Scholar , 2 WHOWorld malaria report 2020: 20 years of global progress and challenges. https://www.who.int/publications/i/item/9789240015791Date: Nov 30, 2020 Date accessed: December 5, 2021 Google Scholar Between 2000 and 2019, an estimated 1·5 billion malaria cases and 7·6 million malaria deaths were averted, with many of the early gains attributed to widescale deployment of insecticide-treated nets and availability of effective drugs. 1 Bhatt S Weiss DJ Cameron E et al. The effect of malaria control on Plasmodium falciparum in Africa between 2000 and 2015. Nature. 2015; 526: 207-211 Crossref PubMed Scopus (1439) Google Scholar However, progress in global malaria control and elimination efforts have stagnated in recent years, the reasons for which are multifactorial and include the development and spread of insecticide resistance in malaria vectors. The massive scale-up of insecticide-treated nets and, later, long-lasting insecticidal nets (LLINs), which have an insecticide incorporated into the net fibre and can last longer under typical use, was accompanied by the emergence of vector resistance to pyrethroids. 3 Hancock PA Hendriks CJM Tangena JA et al. Mapping trends in insecticide resistance phenotypes in African malaria vectors. PLoS Biol. 2020; 18e3000633 Crossref PubMed Scopus (37) Google Scholar This resistance is now present in all primary vectors throughout much of sub-Saharan Africa. 3 Hancock PA Hendriks CJM Tangena JA et al. Mapping trends in insecticide resistance phenotypes in African malaria vectors. PLoS Biol. 2020; 18e3000633 Crossref PubMed Scopus (37) Google Scholar , 4 Knox TB Juma EO Ochomo EO et al. An online tool for mapping insecticide resistance in major Anopheles vectors of human malaria parasites and review of resistance status for the Afrotropical region. Parasit Vectors. 2014; 7: 76 Crossref PubMed Scopus (86) Google Scholar , 5 Moyes CL Athinya DK Seethaler T et al. Evaluating insecticide resistance across African districts to aid malaria control decisions. Proc Natl Acad Sci USA. 2020; 117: 22042-22050 Crossref PubMed Scopus (17) Google Scholar Studies have shown that a new generation of LLINs that include the synergist piperonyl butoxide have improved efficacy compared with pyrethroid-only nets. 6 WHOGlobal plan for insecticide resistance management in malaria vectors. https://apps.who.int/iris/handle/10665/44846Date: 2012 Date accessed: December 5, 2021 Google Scholar , 7 Mosha JF Kulkarni MA Lukole E et al. Effectiveness and cost-effectiveness against malaria of three types of dual-active-ingredient long-lasting insecticidal nets compared with pyrethroid-only long-lasting insecticidal nets in an area with pyrethroid-resistant mosquitoes in Tanzania: a four-arm, cluster-randomised trial. Lancet. 2022; 399: 1227-1241 Summary Full Text Full Text PDF PubMed Scopus (3) Google Scholar However, although piperonyl butoxide can restore susceptibility in pyrethroid-resistant mosquito populations wherein the primary resistance mechanism is metabolic detoxification by oxidase enzymes, it has minimal impact on other mechanisms of pyrethroid resistance. Insecticide-treated nets with novel active ingredients and different modes of action are needed. The WHO Global Plan for Insecticide Resistance Management recommends the use of two or more compounds of different insecticide classes to make a single product or formulation as an approach to combating resistance. 6 WHOGlobal plan for insecticide resistance management in malaria vectors. https://apps.who.int/iris/handle/10665/44846Date: 2012 Date accessed: December 5, 2021 Google Scholar The challenge is developing products that are not only effective against pyrethroid-resistant mosquitoes but are also safe to humans and are amenable to incorporation into the net fibres. Effectiveness and cost-effectiveness against malaria of three types of dual-active-ingredient long-lasting insecticidal nets (LLINs) compared with pyrethroid-only LLINs in Tanzania: a four-arm, cluster-randomised trialAfter 2 years, chlorfenapyr LLINs provided significantly better protection than pyrethroid-only LLINs against malaria in an area with pyrethroid-resistant mosquitoes, and the additional cost of these nets would be considerably below plausible cost-effectiveness thresholds ($292–393 per DALY averted). Before scale-up of chlorfenapyr LLINs, resistance management strategies are needed to preserve their effectiveness. Poor textile and active ingredient durability in the piperonyl butoxide and pyriproxyfen LLINs might have contributed to their relative lack of effectiveness compared with standard LLINs. Full-Text PDF Open Access