Impact of insecticide resistance in Anopheles arabiensis on malaria incidence and prevalence in Sudan and the costs of mitigation.

Insecticide-based interventions have contributed to ∼78% of the reduction in the malaria burden in sub-Saharan Africa since 2000. Insecticide resistance in malaria vectors could presage a catastrophic rebound in disease incidence and mortality. A major impediment to the implementation of insecticide resistance management strategies is that evidence of the impact of resistance on malaria disease burden is limited. A cluster randomized trial was conducted in Sudan with pyrethroid-resistant and carbamate-susceptible malaria vectors. Clusters were randomly allocated to receive either long-lasting insecticidal nets (LLINs) alone or LLINs in combination with indoor residual spraying (IRS) with a pyrethroid (deltamethrin) insecticide in the first year and a carbamate (bendiocarb) insecticide in the two subsequent years. Malaria incidence was monitored for 3 y through active case detection in cohorts of children aged 1 to <10 y. When deltamethrin was used for IRS, incidence rates in the LLIN + IRS arm and the LLIN-only arm were similar, with the IRS providing no additional protection [incidence rate ratio (IRR) = 1.0 (95% confidence interval [CI]: 0.36-3.0; P = 0.96)]. When bendiocarb was used for IRS, there was some evidence of additional protection [interaction IRR = 0.55 (95% CI: 0.40-0.76; P < 0.001)]. In conclusion, pyrethroid resistance may have had an impact on pyrethroid-based IRS. The study was not designed to assess whether resistance had an impact on LLINs. These data alone should not be used as the basis for any policy change in vector control interventions.

Characterization and monitoring of deltamethrin-resistance in Anopheles culicifacies in the presence of a long-lasting insecticide-treated net intervention.

BACKGROUND: Deltamethrin-impregnated, long-lasting insecticidal nets (LLINs) were distributed in the study area from November 2014 to January 2015 to evaluate their impact on malaria transmission in the presence of insecticide-resistant vectors. Studies were carried out in 16 selected clusters in Keshkal sub-district, Chhattisgarh State, India to monitor and characterize deltamethrin resistance in Anopheles culicifacies sensu lato. RESULTS: Deltamethrin susceptibility of An. culicifacies decreased in a post-LLIN survey compared to a pre-LLIN survey and was not significant (p > 0.05) while, the knockdown values showed significant increase (p < 0.05). Pre-exposure to piperonyl butoxide, triphenyl phosphate showed synergism against deltamethrin (p < 0.001). Biochemical assays showed significantly (p < 0.05) elevated monooxygenases in 3 of 5 clusters in post-LLIN survey-I that increased to 10 of 11 clusters in post-LLIN survey-II, while esterases were found significantly elevated in all clusters and both enzymes were involved in conferring pyrethroid resistance, not discounting the involvement of kdr (L1014L/S) gene that was heterozygous and at low frequency (4-5%). CONCLUSION: This field study, in a tribal district of India, after distribution of deltamethrin-impregnated LLINs showed decrease in deltamethrin susceptibility in An. culicifacies, a major vector of malaria in this study area and in India. Results indicated development of resistance as imminent with the increase in insecticide selection pressure. There is an urgent need to develop new vector control tools, with insecticide classes having novel mechanisms of resistance, to avoid or delay the onset of resistance. Regular insecticide resistance monitoring and mechanistic studies should be the priority for the malaria control programmes to suggest strategies for insecticide resistance management. The global commitment to eliminate malaria by 2030 needs various efforts that include development of combination vector control products and interventions and few are becoming available.

Insecticide-Treated Nets and Protection against Insecticide-Resistant Malaria Vectors in Western Kenya.

Insecticide resistance might reduce the efficacy of malaria vector control. In 2013 and 2014, malaria vectors from 50 villages, of varying pyrethroid resistance, in western Kenya were assayed for resistance to deltamethrin. Long-lasting insecticide-treated nets (LLIN) were distributed to households at universal coverage. Children were recruited into 2 cohorts, cleared of malaria-causing parasites, and tested every 2 weeks for reinfection. Infection incidence rates for the 2 cohorts were 2.2 (95% CI 1.9-2.5) infections/person-year and 2.8 (95% CI 2.5-3.0) infections/person-year. LLIN users had lower infection rates than non-LLIN users in both low-resistance (rate ratio 0.61, 95% CI 0.42-0.88) and high-resistance (rate ratio 0.55, 95% CI 0.35-0.87) villages (p = 0.63). The association between insecticide resistance and infection incidence was not significant (p = 0.99). Although the incidence of infection was high among net users, LLINs provided significant protection (p = 0.01) against infection with malaria parasite regardless of vector insecticide resistance.

A significant association between deltamethrin resistance, Plasmodium falciparum infection and the Vgsc-1014S resistance mutation in Anopheles gambiae highlights the epidemiological importance of resistance markers.

BACKGROUND: The success of malaria vector control is threatened by widespread pyrethroid insecticide resistance. However, the extent to which insecticide resistance impacts transmission is unclear. The objective of this study was to examine the association between the DDT/pyrethroid knockdown resistance mutation Vgsc-1014S, commonly termed kdr, and infection with Plasmodium falciparum sporozoites in Anopheles gambiae. METHODS: WHO standard methods were used to characterize susceptibility of wild female mosquitoes to 0.05 % deltamethrin. PCR-based molecular diagnostics were used to identify mosquitoes to species and to genotype at the Vgsc-L1014S locus. ELISAs were used to detect the presence of P. falciparum sporozoites and for blood meal identification. RESULTS: Anopheles mosquitoes were resistant to deltamethrin with mortality rates of 77.7 % [95 % CI 74.9-80.3 %]. Of 545 mosquitoes genotyped 96.5 % were A. gambiae s.s. and 3.5 % were Anopheles arabiensis. The Vgsc-1014S mutation was detected in both species. Both species were predominantly anthropophagic. In A. gambiae s.s., Vgsc-L1014S genotype was significantly associated with deltamethrin resistance (χ2 = 11.2; p < 0.001). The P. falciparum sporozoite infection rate was 4.2 %. There was a significant association between the presence of sporozoites and Vgsc-L1014S genotype in A. gambiae s.s. (χ2 = 4.94; p = 0.026). CONCLUSIONS: One marker, Vgsc-1014S, was associated with insecticide resistance and P. falciparum infection in wild-caught mixed aged populations of A. gambiae s.s. thereby showing how resistance may directly impact transmission.

Does insecticide resistance contribute to heterogeneities in malaria transmission in The Gambia?

BACKGROUND: Malaria hotspots, areas with consistently higher than average transmission, may become increasingly common as malaria declines. This phenomenon, currently observed in The Gambia, may be caused by several factors, including some related to the local vectors, whose contribution is poorly understood. METHODS: Using WHO susceptibility bioassays, insecticide resistance status was determined in vector populations sampled from six pairs of villages across The Gambia, each pair contained a low and high prevalence village. RESULTS: Three vector species were observed (23.5% Anopheles arabiensis, 31.2% Anopheles gambiae, 43.3% Anopheles coluzzii and 2.0% An. coluzzii × An. gambiae hybrids). Even at a fine scale, significant differences in species composition were detected within village pairs. Resistance to both DDT and deltamethrin was more common in An. gambiae, most markedly in the eastern part of The Gambia and partly attributable to differing frequencies of resistance mutations. The Vgsc-1014F target site mutation was strongly associated with both DDT (OR = 256.7, (95% CI 48.6-6374.3, p < 0.001) and deltamethrin survival (OR = 9.14, (95% CI 4.24-21.4, p < 0.001). A second target site mutation, Vgsc-1575Y, which co-occurs with Vgsc-1014F, and a metabolic marker of resistance, Gste2-114T, conferred additional survival benefits to both insecticides. DDT resistance occurred significantly more frequently in villages with high malaria prevalence (p = 0.025) though this did not apply to deltamethrin resistance. CONCLUSION: Whilst causality of relationships requires further investigation, variation in vector species and insecticide resistance in The Gambia is associated with malaria endemicity; with a notably higher prevalence of infection and insecticide resistance in the east of the country. In areas with heterogeneous malaria transmission, the role of the vector should be investigated to guide malaria control interventions.

Design of a study to determine the impact of insecticide resistance on malaria vector control: a multi-country investigation.

BACKGROUND: Progress in reducing the malaria disease burden through the substantial scale up of insecticide-based vector control in recent years could be reversed by the widespread emergence of insecticide resistance. The impact of insecticide resistance on the protective effectiveness of insecticide-treated nets (ITN) and indoor residual spraying (IRS) is not known. A multi-country study was undertaken in Sudan, Kenya, India, Cameroon and Benin to quantify the potential loss of epidemiological effectiveness of ITNs and IRS due to decreased susceptibility of malaria vectors to insecticides. The design of the study is described in this paper. METHODS: Malaria disease incidence rates by active case detection in cohorts of children, and indicators of insecticide resistance in local vectors were monitored in each of approximately 300 separate locations (clusters) with high coverage of malaria vector control over multiple malaria seasons. Phenotypic and genotypic resistance was assessed annually. In two countries, Sudan and India, clusters were randomly assigned to receive universal coverage of ITNs only, or universal coverage of ITNs combined with high coverage of IRS. Association between malaria incidence and insecticide resistance, and protective effectiveness of vector control methods and insecticide resistance were estimated, respectively. RESULTS: Cohorts have been set up in all five countries, and phenotypic resistance data have been collected in all clusters. In Sudan, Kenya, Cameroon and Benin data collection is due to be completed in 2015. In India data collection will be completed in 2016. DISCUSSION: The paper discusses challenges faced in the design and execution of the study, the analysis plan, the strengths and weaknesses, and the possible alternatives to the chosen study design.

Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae.

Insecticide resistance is an ideal model to study the emergence and spread of adaptative variants. In the African malaria mosquito, Anopheles gambiae, this is complemented by a strong public health rationale. In this insect, resistance to pyrethroid and DDT insecticides is strongly associated with the mutations L1014F and L1014S within the para voltage-gated sodium channel (VGSC). Across much of West Africa, 1014F frequency approaches fixation. Here, we document the emergence of a mutation, N1575Y, within the linker between domains III-IV of the VGSC. In data extending over 40 kbp of the VGSC 1575Y occurs on only a single long-range haplotype, also bearing 1014F. The 1014F-1575Y haplotype was found in both M and S molecular forms of An. gambiae in West/Central African sample sites separated by up to 2,000 km. In Burkina Faso M form, 1575Y allele frequency rose significantly from 0.053 to 0.172 between 2008 and 2010. Extended haplotype homozygosity analysis of the wild-type 1575N allele showed rapid decay of linkage disequilibrium (LD), in sharp contrast to the extended LD exhibited by 1575Y. A haplotype with long-range LD and high/increasing frequency is a classical sign of strong positive selection acting on a recent mutant. 1575Y occurs ubiquitously on a 1014F haplotypic background, suggesting that the N1575Y mutation compensates for deleterious fitness effects of 1014F and/or confers additional resistance to insecticides. Haplotypic tests of association suggest the latter: The 1014F-1575Y haplotype confers a significant additive benefit above 1014F-1575N for survival to DDT (M form P = 0.03) and permethrin (S form P = 0.003).

Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana.

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Cryptic population structure and insecticide resistance in Anopheles gambiae from the southern Democratic Republic of Congo.

The Democratic Republic of Congo (DRC) suffers from one of the highest malaria burdens worldwide, but information on its Anopheles vector populations is relatively limited. Preventative malaria control in DRC is reliant on pyrethroid-treated nets, raising concerns over the potential impacts of insecticide resistance. We sampled Anopheles gambiae from three geographically distinct populations (Kimpese, Kapolowe and Mikalayi) in southern DRC, collecting from three sub-sites per population and characterising mosquito collections from each for resistance to pyrethroids using WHO tube bioassays. Resistance to each of three different pyrethroids was generally high in An. gambiae with < 92% mortality in all tests, but varied between collections, with mosquitoes from Kimpese being the most resistant. Whole genome sequencing of 165 An. gambiae revealed evidence for genetic differentiation between Kimpese and Kapolowe/Mikalayi, but not between the latter two sample sites despite separation of approximately 800 km. Surprisingly, there was evidence of population structure at a small spatial scale between collection subsites in Kimpese, despite separation of just tens of kilometres. Intra-population (H12) and inter-population (FST) genome scans identified multiple peaks corresponding to genes associated with insecticide resistance such as the voltage gated sodium channel (Vgsc) target site on chromosome 2L, a Cyp6 cytochrome P450 cluster on chromosome arm 2R, and the Cyp9k1 P450 gene on chromosome X. In addition, in the Kimpese subsites, the P450 redox partner gene Cpr showed evidence for contemporary selection (H12) and population differentiation (FST) meriting further exploration as a potential resistance associated marker.

Spatiotemporal distribution and bionomics of Anopheles stephensi in different eco-epidemiological settings in Ethiopia.

BACKGROUND: Malaria is a major public health concern in Ethiopia, and its incidence could worsen with the spread of the invasive mosquito species Anopheles stephensi in the country. This study aimed to provide updates on the distribution of An. stephensi and likely household exposure in Ethiopia. METHODS: Entomological surveillance was performed in 26 urban settings in Ethiopia from 2021 to 2023. A kilometer-by-kilometer quadrant was established per town, and approximately 20 structures per quadrant were surveyed every 3 months. Additional extensive sampling was conducted in 50 randomly selected structures in four urban centers in 2022 and 2023 to assess households' exposure to An. stephensi. Prokopack aspirators and CDC light traps were used to collect adult mosquitoes, and standard dippers were used to collect immature stages. The collected mosquitoes were identified to species level by morphological keys and molecular methods. PCR assays were used to assess Plasmodium infection and mosquito blood meal source. RESULTS: Catches of adult An. stephensi were generally low (mean: 0.15 per trap), with eight positive sites among the 26 surveyed. This mosquito species was reported for the first time in Assosa, western Ethiopia. Anopheles stephensi was the predominant species in four of the eight positive sites, accounting for 75-100% relative abundance of the adult Anopheles catches. Household-level exposure, defined as the percentage of households with a peridomestic presence of An. stephensi, ranged from 18% in Metehara to 30% in Danan. Anopheles arabiensis was the predominant species in 20 of the 26 sites, accounting for 42.9-100% of the Anopheles catches. Bovine blood index, ovine blood index and human blood index values were 69.2%, 32.3% and 24.6%, respectively, for An. stephensi, and 65.4%, 46.7% and 35.8%, respectively, for An. arabiensis. None of the 197 An. stephensi mosquitoes assayed tested positive for Plasmodium sporozoite, while of the 1434 An. arabiensis mosquitoes assayed, 62 were positive for Plasmodium (10 for P. falciparum and 52 for P. vivax). CONCLUSIONS: This study shows that the geographical range of An. stephensi has expanded to western Ethiopia. Strongly zoophagic behavior coupled with low adult catches might explain the absence of Plasmodium infection. The level of household exposure to An. stephensi in this study varied across positive sites. Further research is needed to better understand the bionomics and contribution of An. stephensi to malaria transmission.

Patterns of Kdr-L995F Allele Emergence Alongside Detoxifying Enzymes Associated with Deltamethrin Resistance in Anopheles gambiae s.l. from North Cameroon.

Understanding how multiple insecticide resistance mechanisms occur in malaria vectors is essential for efficient vector control. This study aimed at assessing the evolution of metabolic mechanisms and Kdr L995F/S resistance alleles in Anopheles gambiae s.l. from North Cameroon, following long-lasting insecticidal nets (LLINs) distribution in 2011. Female An. gambiae s.l. emerging from larvae collected in Ouro-Housso/Kanadi, Be-Centre, and Bala in 2011 and 2015, were tested for susceptibility to deltamethrin + piperonyl butoxide (PBO) or SSS-tributyl-phosphoro-thrithioate (DEF) synergists, using the World Health Organization's standard protocol. The Kdr L995F/S alleles were genotyped using Hot Ligation Oligonucleotide Assay. Tested mosquitoes identified using PCR-RFLP were composed of An. arabiensis (68.5%), An. coluzzii (25.5%) and An. gambiae (6%) species. From 2011 to 2015, metabolic resistance increased in Ouro-Housso/Kanadi (up to 89.5% mortality to deltametnrin+synergists in 2015 versus <65% in 2011; p < 0.02), while it decreased in Be-Centre and Bala (>95% mortality in 2011 versus 42-94% in 2015; p < 0.001). Conversely, the Kdr L995F allelic frequencies slightly decreased in Ouro-Housso/Kanadi (from 50% to 46%, p > 0.9), while significantly increasing in Be-Centre and Bala (from 0-13% to 18-36%, p < 0.02). These data revealed two evolutionary trends of deltamethrin resistance mechanisms; non-pyrethroid vector control tools should supplement LLINs in North Cameroon.

Transcriptomic analysis of insecticide resistance in the lymphatic filariasis vector Culex quinquefasciatus.

Culex quinquefasciatus plays an important role in transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF), as well as many arboviral diseases. Currently, efforts to tackle C. quinquefasciatus vectored diseases are based on either mass drug administration (MDA) for LF, or insecticide-based interventions. Widespread and intensive insecticide usage has resulted in increased resistance in mosquito vectors, including C. quinquefasciatus. Herein, the transcriptome profile of Ugandan bendiocarb-resistant C. quinquefasciatus was explored to identify candidate genes associated with insecticide resistance. High levels of insecticide resistance were observed for five out of six insecticides tested, with the lowest mortality (0.97%) reported to permethrin, while for DDT, lambdacyhalothrin, bendiocarb and deltamethrin the mortality rate ranged from 1.63-3.29%. Resistance to bendiocarb in exposed mosquitoes was marked, with 2.04% mortality following 1 h exposure and 58.02% after 4 h. Genotyping of the G119S Ace-1 target site mutation detected a highly significant association (p < 0.0001; OR = 25) between resistance and Ace1-119S. However, synergist assays using the P450 inhibitor PBO, or the esterase inhibitor TPP resulted in markedly increased mortality (to ≈80%), suggesting a role of metabolic resistance in the resistance phenotype. Using a novel, custom 60 K whole-transcriptome microarray 16 genes significantly overexpressed in resistant mosquitoes were detected, with the P450 Cyp6z18 showing the highest differential gene expression (>8-fold increase vs unexposed controls). These results provide evidence that bendiocarb resistance in Ugandan C. quinquefasciatus is mediated by both target-site mechanisms and over-expression of detoxification enzymes.

Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus.

BACKGROUND: Culex quinquefasciatus has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda. RESULTS: This new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches, pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with pyrosequencing and TaqMan results (98.64% and 100% respectively). CONCLUSIONS: Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

Spatial and temporal development of deltamethrin resistance in malaria vectors of the Anopheles gambiae complex from North Cameroon.

The effectiveness of insecticide-based malaria vector control interventions in Africa is threatened by the spread and intensification of pyrethroid resistance in targeted mosquito populations. The present study aimed at investigating the temporal and spatial dynamics of deltamethrin resistance in An. gambiae s.l. populations from North Cameroon. Mosquito larvae were collected from 24 settings of the Garoua, Pitoa and Mayo Oulo Health Districts (HDs) from 2011 to 2015. Two to five days old female An. gambiae s.l. emerging from larval collections were tested for deltamethrin resistance using the World Health Organization's (WHO) standard protocol. Sub samples of test mosquitoes were identified to species using PCR-RFLP and genotyped for knockdown resistance alleles (Kdr 1014F and 1014S) using Hot Ligation Oligonucleotide Assay (HOLA). All the tested mosquitoes were identified as belonging to the An. gambiae complex, including 3 sibling species mostly represented by Anopheles arabiensis (67.6%), followed by Anopheles coluzzii (25.4%) and Anopheles gambiae (7%). Deltamethrin resistance frequencies increased significantly between 2011 and 2015, with mosquito mortality rates declining from 70-85% to 49-73% in the three HDs (Jonckheere-Terstra test statistic (JT) = 5638, P< 0.001), although a temporary increase of mortality rates (91-97%) was seen in the Pitoa and Mayo Oulo HDs in 2012. Overall, confirmed resistance emerged in 10 An. gambiae s.l. populations over the 24 field populations monitored during the study period, from 2011 to 2015. Phenotypic resistance was mostly found in urban settings compared with semi-urban and rural settings (JT = 5282, P< 0.0001), with a spatial autocorrelation between neighboring localities. The Kdr 1014F allelic frequencies in study HDs increased from 0-30% in 2011 to 18-61% in 2014-2015 (JT = 620, P <0.001), especially in An. coluzzii samples. The overall frequency of the Kdr 1014S allele was 0.1%. This study revealed a rapid increase and widespread deltamethrin resistance frequency as well as Kdr 1014F allelic frequencies in An. gambiae s.l. populations over time, emphasizing the urgent need for vector surveillance and insecticide resistance management strategies in Cameroon.

Implications of insecticide resistance for malaria vector control with long-lasting insecticidal nets: a WHO-coordinated, prospective, international, observational cohort study.

BACKGROUND: Scale-up of insecticide-based interventions has averted more than 500 million malaria cases since 2000. Increasing insecticide resistance could herald a rebound in disease and mortality. We aimed to investigate whether insecticide resistance was associated with loss of effectiveness of long-lasting insecticidal nets and increased malaria disease burden. METHODS: This WHO-coordinated, prospective, observational cohort study was done at 279 clusters (villages or groups of villages in which phenotypic resistance was measurable) in Benin, Cameroon, India, Kenya, and Sudan. Pyrethroid long-lasting insecticidal nets were the principal form of malaria vector control in all study areas; in Sudan this approach was supplemented by indoor residual spraying. Cohorts of children from randomly selected households in each cluster were recruited and followed up by community health workers to measure incidence of clinical malaria and prevalence of infection. Mosquitoes were assessed for susceptibility to pyrethroids using the standard WHO bioassay test. Country-specific results were combined using meta-analysis. FINDINGS: Between June 2, 2012, and Nov 4, 2016, 40 000 children were enrolled and assessed for clinical incidence during 1·4 million follow-up visits. 80 000 mosquitoes were assessed for insecticide resistance. Long-lasting insecticidal net users had lower infection prevalence (adjusted odds ratio [OR] 0·63, 95% CI 0·51-0·78) and disease incidence (adjusted rate ratio [RR] 0·62, 0·41-0·94) than did non-users across a range of resistance levels. We found no evidence of an association between insecticide resistance and infection prevalence (adjusted OR 0·86, 0·70-1·06) or incidence (adjusted RR 0·89, 0·72-1·10). Users of nets, although significantly better protected than non-users, were nevertheless subject to high malaria infection risk (ranging from an average incidence in net users of 0·023, [95% CI 0·016-0·033] per person-year in India, to 0·80 [0·65-0·97] per person year in Kenya; and an average infection prevalence in net users of 0·8% [0·5-1·3] in India to an average infection prevalence of 50·8% [43·4-58·2] in Benin). INTERPRETATION: Irrespective of resistance, populations in malaria endemic areas should continue to use long-lasting insecticidal nets to reduce their risk of infection. As nets provide only partial protection, the development of additional vector control tools should be prioritised to reduce the unacceptably high malaria burden. FUNDING: Bill & Melinda Gates Foundation, UK Medical Research Council, and UK Department for International Development.

Detection and quantitation of copy number variation in the voltage-gated sodium channel gene of the mosquito Culex quinquefasciatus.

Insecticide resistance is typically associated with alterations to the insecticidal target-site or with gene expression variation at loci involved in insecticide detoxification. In some species copy number variation (CNV) of target site loci (e.g. the Ace-1 target site of carbamate insecticides) or detoxification genes has been implicated in the resistance phenotype. We show that field-collected Ugandan Culex quinquefasciatus display CNV for the voltage-gated sodium channel gene (Vgsc), target-site of pyrethroid and organochlorine insecticides. In order to develop field-applicable diagnostics for Vgsc CN, and as a prelude to investigating the possible association of CN with insecticide resistance, three assays were compared for their accuracy in CN estimation in this species. The gold standard method is droplet digital PCR (ddPCR), however, the hardware is prohibitively expensive for widespread utility. Here, ddPCR was compared to quantitative PCR (qPCR) and pyrosequencing. Across all platforms, CNV was detected in ≈10% of mosquitoes, corresponding to three or four copies (per diploid genome). ddPCR and qPCR-Std-curve yielded similar predictions for Vgsc CN, indicating that the qPCR protocol developed here can be applied as a diagnostic assay, facilitating monitoring of Vgsc CN in wild populations and the elucidation of association between the Vgsc CN and insecticide resistance.

Local selection in the presence of high levels of gene flow: Evidence of heterogeneous insecticide selection pressure across Ugandan Culex quinquefasciatus populations.

BACKGROUND: Culex quinquefasciatus collected in Uganda, where no vector control interventions directly targeting this species have been conducted, was used as a model to determine if it is possible to detect heterogeneities in selection pressure driven by insecticide application targeting other insect species. METHODOLOGY/PRINCIPAL FINDINGS: Population genetic structure was assessed through microsatellite analysis, and the impact of insecticide pressure by genotyping two target-site mutations, Vgsc-1014F of the voltage-gated sodium channel target of pyrethroid and DDT insecticides, and Ace1-119S of the acetylcholinesterase gene, target of carbamate and organophosphate insecticides. No significant differences in genetic diversity were observed among populations by microsatellite markers with HE ranging from 0.597 to 0.612 and low, but significant, genetic differentiation among populations (FST = 0.019, P = 0.001). By contrast, the insecticide-resistance markers display heterogeneous allelic distributions with significant differences detected between Central Ugandan (urban) populations relative to Eastern and Southwestern (rural) populations. In the central region, a frequency of 62% for Vgsc-1014F, and 32% for the Ace1-119S resistant allele were observed. Conversely, in both Eastern and Southwestern regions the Vgsc-1014F alleles were close to fixation, whilst Ace1-119S allele frequency was 12% (although frequencies may be underestimated due to copy number variation at both loci). CONCLUSIONS/SIGNIFICANCE: Taken together, the microsatellite and both insecticide resistance target-site markers provide evidence that in the face of intense gene flow among populations, disjunction in resistance frequencies arise due to intense local selection pressures despite an absence of insecticidal control interventions targeting Culex.

Evolution of insecticide resistance diagnostics in malaria vectors.

Malaria control is reliant upon effective, programmatic-scale, anti-vector interventions. The widespread distribution of pyrethroid-treated bednets in sub-Saharan Africa has been a driver of morbidity and mortality reductions over the last decade. Unfortunately resistance to insecticides, and to pyrethroids in particular, is increasingly common in Anopheles malaria vectors, and is a major threat to continued control and future elimination. Here we argue that current methods to diagnose resistance often have limited utility and should be augmented or even partially replaced by wider application of DNA markers.

Pyrethroid susceptibility of malaria vectors in four Districts of western Kenya.

BACKGROUND: Increasing pyrethroid resistance in malaria vectors has been reported in western Kenya where long lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the mainstays of vector control. To ensure the sustainability of insecticide-based malaria vector control, monitoring programs need to be implemented. This study was designed to investigate the extent and distribution of pyrethroid resistance in 4 Districts of western Kenya (Nyando, Rachuonyo, Bondo and Teso). All four Districts have received LLINs while Nyando and Rachuonyo Districts have had IRS campaigns for 3-5 years using pyrethroids. This study is part of a programme aimed at determining the impact of insecticide resistance on malaria epidemiology. METHODS: Three day old adult mosquitoes from larval samples collected in the field, were used for bioassays using the WHO tube bioassay, and mortality recorded 24 hours post exposure. Resistance level was assigned based on the 2013 WHO guidelines where populations with <90% mortality were considered resistant. Once exposed, samples were identified to species using PCR. RESULTS: An. arabiensis comprised at least 94% of all An. gambiae s.l. in Bondo, Rachuonyo and Nyando. Teso was a marked contrast case with 77% of all samples being An. gambiae s.s. Mortality to insecticides varied widely between clusters even in one District with mortality to deltamethrin ranging from 45-100%, while to permethrin the range was 30-100%. Mortality to deltamethrin in Teso District was < 90% in 4 of 6 clusters tested in An arabiensis and <90% in An. gambiae s.s in 5 of 6 clusters tested. To permethrin, mortality ranged between 5.9-95%, with <90% mortality in 9 of 13 and 8 of 13 in An. arabiensis and An. gambiae s.s. respectively. Cluster specific mortality of An. arabiensis between permethin and deltamethrin were not correlated (Z = 2.9505, P = 0.2483). CONCLUSION: High levels of pyrethroid resistance were observed in western Kenya. This resistance does not seem to be associated with either species or location. Insecticide resistance can vary within small geographical areas and such heterogeneity may make it possible to evaluate the impact of resistance on malaria and mosquito parameters within similar eco-epidemiological zones.