BackgroundMutations in the voltage-gated sodium channel at codon 1014 confer knock-down resistance (kdr) to pyrethroids in a wide range of insects. Anopheles gambiae exhibits two mutant alleles at codon 1014, serine and phenylalanine; and both are now widespread across Africa. Existing screening methods only allow for one resistant allele to be detected per assay. A new locked nucleic acid (LNA) qPCR assay was developed for the simultaneous detection of both mutant alleles and the wild type allele in a single assay. This tri-allelic detection assay was assessed as part of a study of the insecticide resistance in An. gambiae sensu stricto (s.s.) in the previously un-sampled area of Nord Ubangi, Democratic Republic of the Congo.MethodsSamples from three sites were tested for insecticide susceptibility using WHO bioassays, with and without the synergist PBO preceding pyrethroid exposures, and were subsequently analysed for frequency and resistance-association of the Vgsc-1014 and Vgsc-N1575Y mutations. Results from the LNA-kdr 1014 assay were compared to results from standard TaqMan-kdr assays.ResultsAnopheles gambiae sensu lato (s.l.) was by far the predominant vector captured (84%), with only low frequencies of Anopheles funestus s.l. (9%) detected in Nord Ubangi. Molecular identification found An. gambiae s.s. to be the principal vector (99%) although Anopheles coluzzii was detected at very low frequency. Anopheles gambiae were susceptible to the carbamate insecticide bendiocarb, but resistant to DDT and to the pyrethroids permethrin and deltamethrin. Susceptibility to both pyrethroids was partially restored with prior exposure to PBO suggesting likely involvement of metabolic resistance. Anopheles gambiae s.s. was homozygous for kdr resistant alleles with both the L1014F and L1014S mutations present, and the N1575Y polymorphism was present at low frequency. The LNA-kdr assay simultaneously detected both resistant alleles and gave results entirely consistent with those from the two TaqMan-kdr assays.ConclusionThis study provides rare data on insecticide resistance and mechanisms in Anopheles from the centre of Africa, with the first detection of N1575Y. Nord Ubangi populations of An. gambiae s.s. show insecticide resistance mediated by both metabolic mechanisms and Vgsc mutations. The LNA-kdr assay is particularly suitable for use in populations in which both 1014S and 1014F kdr alleles co-occur and provides robust results, with higher throughput and at a quarter of the cost of TaqMan assays.Electronic supplementary materialThe online version of this article (10.1186/s12936-018-2561-5) contains supplementary material, which is available to authorized users.

BackgroundLong-lasting insecticidal nets (LLINs) are the principal tool for malaria control in Africa and are presently treated with a single class of insecticide; however, increasing levels of insecticide resistance threaten their success. In response to this threat nets have been developed that incorporate the synergist, piperonyl butoxide (PBO), which inhibits the activity of cytochrome P450s which is one main mechanisms of insecticide resistance, allowing resistance to pyrethroids to be reversed. However, data on the value and cost effectiveness of these nets is lacking. A large-scale cluster randomised trial of conventional LLINs and PBO-LLINs was conducted in Uganda in 104 health sub-districts (HSDs) in 2017–2019. Prior to the mass distribution of LLINs, a baseline entomological survey was carried out, the results of which are reported herein. Ten households from each HSD were randomly selected for entomological surveillance at baseline which included household mosquito collections.ResultsPrior to LLIN distribution entomological collections were carried out in 1029 houses across the 104 HSDs. Anopheles gambiae (s.l.) was the principal vector in all but 9 of the 71 HSDs that yielded vector species. Molecular analysis found An. gambiae (s.s.) to be the predominant vector collected. Plasmodium falciparum was detected in 5.5% of An. gambiae (s.s.) and in 4.0% of An. funestus (s.s.) examined. Infection rates of other plasmodium species (P. vivax, P. ovale and P. malariae) were lower with infection rates of 1.2% and 1.7% for An. gambiae (s.s.) and An. funestus (s.s.), respectively. The knockdown resistance (kdr) mutation Vgsc-L1014S was found at very high frequency in An. gambiae (s.s.) with the Vgsc-L1014F mutation at low frequency and the wild-type allele virtually absent. In An. arabiensis the wild-type allele was predominant. The resistance-associated alleles, Cyp4j5-L43F and Coeae1d were found at moderate frequencies which varied across the study site. Vgsc-N1575Y mutation was not found in any samples examined.ConclusionsNo significant differences between planned intervention arms was observed in vector densities, sporozoite infection rate or insecticide resistance marker frequency across the study site prior to the distribution of LLINs. Very high levels of kdr resistance were observed in all areas; however, the resistance-associated markers Cyp4j5-L43F and Coeae1d were found at varying frequencies across the study site which may have implications for the effectiveness of standard LLINs.Trial registration This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395Electronic supplementary materialThe online version of this article (10.1186/s13071-019-3353-7) contains supplementary material, which is available to authorized users.

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistancebreaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae

Insecticide resistance provides both an increasingly pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on deployment of pyrethroid insecticides, primarily in long lasting insecticide treated nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years in concert with dramatic expansion of LLIN distributions. Identifying genetic mechanisms causing high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000G) data we identified a very recent selective sweep in Ugandan mosquitoes which localized to a cluster of cytochrome P450 genes, including some frequently implicated in resistance. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region around lake Victoria, with stepwise replacement of the double-mutant (Zanzibar TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids; is strongly predictive of resistance to pyrethroids most notably deltamethrin, a commonly-used LLIN insecticide, but importantly, appears less effective against LLINs co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable even within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries. Duplications of Cyp6aa1 gene are common in An. gambiae across Africa and are likely to be a useful diagnostic for high levels of pyrethroid resistance.

Insecticide resistance provides both a pressing threat to the control of vector-borne diseases and insights into the remarkable capacity of natural populations to show rapid evolutionary responses. Malaria control remains heavily dependent on deployment of insecticides, primarily in long lasting insecticide treated nets (LLINs), but resistance in the major malaria vectors has increased over the last 15 years. Identifying genetic mechanisms causing high-level resistance in mosquitoes, which may almost entirely overcome pyrethroid efficacy, is crucial for the development and deployment of potentially resistance-breaking tools. Using the Anopheles gambiae 1000 genomes data we identified a very recent selective sweep in Ugandan mosquitoes which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a point mutation in Cyp6p4, an insertion of a partial Zanzibar transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border region, with stepwise replacement of the double-mutant (Zanzibar TE and Cyp6p4-236M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolise pyrethroids; is strongly predictive of resistance to pyrethroids but importantly, appears less effective against LLINs co-treated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable even within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid co-treated LLINs across African countries.

Long Lasting Insecticidal Nets (LLINs) provide physical and chemical protection from the bites of malaria transmitting mosquitoes but growing resistance to pyrethroid insecticides threatens their effectiveness. To restore insecticidal effect, pyrethroid LLINs supplemented with the synergist piperonyl butoxide (PBO) have been developed. However, the durability of these new products in the field must be assessed. In 2017-2018 the Ugandan Ministry of Health undertook a mass-distribution of LLINs with and without PBO, providing an opportunity to assess durability. A pragmatic cluster-randomised trial was embedded into the 2017-2018 national distribution. A total of 104 clusters were included with each receiving one of four LLIN products, two of which contained pyrethroid and PBO (Olyset Plus and PermaNet 3.0) and two which had pyrethroid only (Olyset Net and PermaNet 2.0). Nets were sampled at baseline, 12 months, and 25 months post-distribution to assess physical condition, chemical content, and bioefficacy. Physical condition was quantified using proportionate Hole Index and total chemical content measured using high-performance liquid chromatography (HPLC). Bioefficacy was assessed by performing three-minute WHO Cone and Wireball assays with pyrethroid-resistant An. gambiae s.s., with 1hr knockdown 24hr mortality recorded. There was no difference in the physical durability of LLINs with PBO and their pyrethroid-only equivalents (p=0.644), with 12.5% of nets in the too torn pHI category after 25 months. However, nets from thatched-roof housing were more likely to be too torn compared to iron-roofed housing (29.7% vs 11.2% respectively, p<0.001). The pyrethroid content of all nets remained relatively stable across timepoints but PBO content declined by 55% (P<0.001) and 58% (p<0.001) for Olyset Plus and PermaNet 3.0 respectively. Both Olyset Plus and PermaNet 3.0 were highly effective against pyrethroid-resistant mosquitoes when new, knocking down 98.93% and 100% respectively. However the bioefficacy of both PBO LLINs declined over time, with Olyset Plus knocking down 45.72% (95% CI: 22.84-68.62, p=0.021) and Permanet 3.0 knocking down 78.57% (95% CI: 63.57-93.58, p<0.001) after 25 months. Both pyrethroid-only LLINs performed poorly in bioassays. Here we demonstrate that both Olyset Plus and PermaNet 3.0 are as durable as their pyrethroid-only equivalents and had superior bioefficacy against pyrethroid-resistant An. gambiae. However, the superiority of PBO-LLINs decreased with operational use, correlating with a reduction in total PBO content. This decline in bioefficacy after just two years is concerning and there is an urgent need to assess the durability of PBO LLINs in other settings. Additionally, the disparity in physical damage between nets collected from traditional and improved housing highlights a need to consider socioeconomic factors when assessing operational lifespan.

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Background Insecticide resistance threatens the effectiveness of malaria vector control, calling for an urgent need to design suitable resistance management strategies. Here, we established the resistance profiling of an Ugandan Anopheles gambiae population to insecticides using WHO procedures and assessed the potential restoration of susceptibility in the hybrid line Mayuge/KISUMU in an insecticide-free environment for eighteen (18) generations. Results This An gambiae population exhibited a very high intensity of resistance to permethrin, deltamethrin, and alphacypermethrin with a consistent loss of efficacy of all long-lasting insecticidal nets (LLINs) tested including PBO-based and new generation nets Interceptor G2 (IG2) and Royal guard. Molecular analysis revealed a fixation of the L1014S-kdr mutation together with the overexpression of some P450 metabolic genes (CYP6Z1, CYP9K1, CYP6P1, 3 & 4) besides the cuticular resistance-related genes (CYP4G16) and sensorial appendage proteins (SAP1, SAP2, and SAP3) but no GSTe2 overexpression. In the absence of selection pressure, the mortality rate after exposure to insecticides increased significantly over generations, and restoration of susceptibility was observed for most of the insecticides in less than 10 generations. Accordingly, a significant reduction in the frequency of KdrE was observed after 13 generations coupled with reduced expression of most metabolic resistance genes. Conclusions The results of this study show that the high intensity of pyrethroid resistance observed in An gambiae from Uganda associated with the loss of efficacy of LLINs could compromise vector control efforts. The study also highlights that an early rotation of insecticides could help manage resistance to insecticides by restoring the susceptibility. However, the persistence of Kdr mutation together with overexpression of some metabolic genes after many generations in the absence of selection pressure indicates the potential implication of modifiers alleviating the cost of resistance which needs to be further investigated.