LLIN Evaluation in Uganda Project (LLINEUP)-effects of a vector control trial on Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Pyrethroid bednets treated with the synergist piperonyl butoxide (PBO) offer the possibility of improved vector control in mosquito populations with metabolic resistance. In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus PBO (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, we conducted cross-sectional household entomological surveys at baseline and then every 6 months for two years, which we use here to investigate longitudinal changes in mosquito infection rate and genetic markers of resistance. Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected (PCR-positive) with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s., but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Distribution of LLINs in Uganda was previously found to be associated with reductions in parasite prevalence and vector density, but here we show that the proportion of infective mosquitoes remained stable across both PBO and non-PBO LLINs, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395 .

Pyrethroid resistance and gene expression profile of a new resistant An. gambiae colony from Uganda reveals multiple resistance mechanisms and overexpression of Glutathione-S-Transferases linked to survival of PBO-pyrethroid combination.

Background: The effectiveness of long-lasting insecticidal nets (LLINs) are being threatened by growing resistance to pyrethroids. To restore their efficacy, a synergist, piperonyl butoxide (PBO) which inhibits cytochrome P450s has been incorporated into pyrethroid treated nets. A trial of PBO-LLINs was conducted in Uganda from 2017 and we attempted to characterize mechanisms of resistance that could impact intervention efficacy. Methods: We established an Anopheles gambiae s.s colony in 2018 using female mosquitoes collected from Busia district in eastern Uganda. We first assessed the phenotypic resistance profile of this colony using WHO tube and net assays using a deltamethrin dose-response approach. The Busia colony was screened for known resistance markers and RT-qPCR targeting 15 genes previously associated with insecticide resistance was performed. Results: The Busia colony had very high resistance to deltamethrin, permethrin and DDT. In addition, the colony had moderate resistance to alpha-cypermethrin and lambda-cyhalothrin but were fully susceptible to bendiocarb and fenitrothion. Exposure to PBO in combination with permethrin and deltamethrin resulted in higher mortality rates in both net and tube assays, with a higher mortality observed in net assays than tube assays. The kdr marker, Vgsc-995S was at very high frequency (91.7-98.9%) whilst the metabolic markers Coeae1d and Cyp4j5-L43F were at very low (1.3% - 11.5%) and moderate (39.5% - 44.7%) frequencies respectively. Our analysis showed that gene expression pattern in mosquitoes exposed to deltamethrin, permethrin or DDT only were similar in comparison to the susceptible strain and there was significant overexpression of cytochrome P450s, glutathione-s-transferases (GSTs) and carboxyl esterases (COEs). However, mosquitoes exposed to both PBO and pyrethroid strikingly and significantly only overexpressed closely related GSTs compared to unexposed mosquitoes while major cytochrome P450s were underexpressed. Conclusions: The high levels of pyrethroid resistance observed in Busia appears associated with a wide range of metabolic gene families.

LLIN Evaluation in Uganda Project (LLINEUP) - Plasmodium infection prevalence and genotypic markers of insecticide resistance in Anopheles vectors from 48 districts of Uganda.

Background: In 2017-2019, we conducted a large-scale, cluster-randomised trial (LLINEUP) to evaluate long-lasting insecticidal nets (LLINs) treated with a pyrethroid insecticide plus the synergist piperonyl butoxide (PBO LLINs), as compared to conventional, pyrethroid-only LLINs across 104 health sub-districts (HSDs) in Uganda. In LLINEUP, and similar trials in Tanzania, PBO LLINs were found to provide greater protection against malaria than conventional LLINs, reducing parasitaemia and vector density. In the LLINEUP trial, cross-sectional entomological surveys were carried out at baseline and then every 6 months for two years. In each survey, ten households per HSD were randomly selected for indoor household entomological collections. Results: Overall, 5395 female Anopheles mosquitoes were collected from 5046 households. The proportion of mosquitoes infected with Plasmodium falciparum did not change significantly over time, while infection with non-falciparum malaria decreased in An. gambiae s.s, but not An. funestus. The frequency of genetic markers associated with pyrethroid resistance increased significantly over time, but the rate of change was not different between the two LLIN types. The knock-down resistance (kdr) mutation Vgsc-995S declined over time as Vgsc-995F, the alternative resistance mutation at this codon, increased. Vgsc-995F appears to be spreading into Uganda. Conclusions: Distribution of LLINs in Uganda was associated with reductions in parasite prevalence and vector density, but the proportion of infective mosquitoes remained stable, suggesting that the potential for transmission persisted. The increased frequency of markers of pyrethroid resistance indicates that LLIN distribution favoured the evolution of resistance within local vectors and highlights the potential benefits of resistance management strategies.Trial registration:: This study is registered with ISRCTN, ISRCTN17516395. Registered 14 February 2017, http://www.isrctn.com/ISRCTN17516395.

Widespread occurrence of copy number variants and fixation of pyrethroid target site resistance in Anopheles gambiae (s.l.) from southern Côte d'Ivoire.

Resistance to pyrethroid and organophosphate insecticides in the malaria vector Anopheles gambiae (s.l.) is conferred by a variety of genetic mutations, including single nucleotide polymorphisms (SNPs) and copy number variants (CNVs). Knowledge of the distribution of these mutations in mosquito populations is a prerequisite for establishing better strategies for their management. In this study, a total of 755 Anopheles gambiae (s.l.) from southern Côte d'Ivoire were exposed to deltamethrin or pirimiphos-methyl insecticides and were screened to assess the distribution of SNPs and CNVs known or believed to confer resistance to one or other of the insecticide classes. Most individuals from the An. gambiae (s.l.) complex were identified by molecular tests as Anopheles coluzzii. Survival to deltamethrin (from 94% to 97%) was higher than to pirimiphos-methyl (from 10% to 49%). In An. gambiae (s.s.), the SNP in the Voltage Gated Sodium Channel (Vgsc) at the 995F locus (Vgsc-995F) was fixed, while other target site mutations were rare or absent (Vgsc-402L: 0%; Vgsc-1570Y: 0%, Acetylcholinesterase Acel-280S: 14%). In An. coluzzii, Vgsc-995F was the target site SNP found at highest frequency (65%) followed by other target site mutations (Vgsc-402L: 36%; Vgsc-1570Y: 0.33%; Acel-280S: 45%). The Vgsc-995S SNP was not present. The presence of the Ace1-280S SNP was found to be significantly linked to the presence of the Ace1-CNV, Ace1_AgDup. Significant association was found between the presence of the Ace1_AgDup and pirimiphos-methyl resistance in An. gambiae (s.s.) but not in An. coluzzii. The deletion Ace1_Del97 was found in one specimen of An. gambiae (s.s.). Four CNVs in the Cyp6aa/Cyp6p gene cluster, which contains genes of known importance for resistance, were detected in An. coluzzii, the most frequent being Dup 7 (42%) and Dup 14 (26%). While none of these individual CNV alleles were significantly associated with resistance, copy number in the Cyp6aa gene region in general was associated with increased resistance to deltamethrin. Elevated expression of Cyp6p3 was nearly associated with deltamethrin resistance, although there was no association of resistance with copy number. Use of alternative insecticides and control methods to arrest resistance spread in An. coluzzii populations is merited.

Characterizing pyrethroid resistance and mechanisms in Anopheles gambiae (s.s.) and Anopheles arabiensis from 11 districts in Uganda.

Insecticide resistance threatens recent progress on malaria control in Africa. To characterize pyrethroid resistance in Uganda, Anopheles gambiae (s.s.) and Anopheles arabiensis were analyzed from 11 sites with varied vector control strategies. Mosquito larvae were collected between May 2018 and December 2020. Sites were categorized as receiving no indoor-residual spraying ('no IRS', n â€‹= â€‹3); where IRS was delivered from 2009 to 2014 and in 2017 and then discontinued ('IRS stopped', n â€‹= â€‹4); and where IRS had been sustained since 2014 ('IRS active', n â€‹= â€‹4). IRS included bendiocarb, pirimiphos methyl and clothianidin. All sites received long-lasting insecticidal nets (LLINs) in 2017. Adult mosquitoes were exposed to pyrethroids; with or without piperonyl butoxide (PBO). Anopheles gambiae (s.s.) and An. arabiensis were identified using PCR. Anopheles gambiae (s.s.) were genotyped for Vgsc-995S/F, Cyp6aa1, Cyp6p4-I236M, ZZB-TE, Cyp4j5-L43F and Coeae1d, while An. arabiensis were examined for Vgsc-1014S/F. Overall, 2753 An. gambiae (s.l.), including 1105 An. gambiae (s.s.) and 1648 An. arabiensis were evaluated. Species composition varied by site; only nine An. gambiae (s.s.) were collected from 'IRS active' sites, precluding species-specific comparisons. Overall, mortality following exposure to permethrin and deltamethrin was 18.8% (148/788) in An. gambiae (s.s.) and 74.6% (912/1222) in An. arabiensis. Mortality was significantly lower in An. gambiae (s.s.) than in An. arabiensis in 'no IRS' sites (permethrin: 16.1 vs 67.7%, P â€‹< â€‹0.001; deltamethrin: 24.6 vs 83.7%, P â€‹< â€‹0.001) and in 'IRS stopped' sites (permethrin: 11.3 vs 63.6%, P â€‹< â€‹0.001; deltamethrin: 25.6 vs 88.9%, P â€‹< â€‹0.001). When PBO was added, mortality increased for An. gambiae (s.s.) and An. arabiensis. Most An. gambiae (s.s.) had the Vgsc-995S/F mutation (95% frequency) and the Cyp6p4-I236M resistance allele (87%), while the frequency of Cyp4j5 and Coeae1d were lower (52% and 55%, respectively). Resistance to pyrethroids was widespread and higher in An. gambiae (s.s.). Where IRS was active, An. arabiensis dominated. Addition of PBO to pyrethroids increased mortality, supporting deployment of PBO LLINs. Further surveillance of insecticide resistance and assessment of associations between genotypic markers and phenotypic outcomes are needed to better understand mechanisms of pyrethroid resistance and to guide vector control.

Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): final results of a cluster-randomised trial embedded in a national distribution campaign.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the foundation of malaria control but resistance of mosquito vectors to pyrethroids threatens their effectiveness. We embedded a cluster-randomised trial into Uganda's 2017-18 campaign to distribute LLINs. LLINs with piperonyl butoxide (PBO) reduced parasite prevalence more effectively than conventional LLINs (without PBO) for 18 months. Here, we report the final 25-month survey results. METHODS: LLINEUP was a cluster-randomised trial conducted in 48 districts in eastern and western Uganda. 104 health subdistricts (clusters) without ongoing or planned indoor residual spraying with pirimiphos-methyl (Actellic, Basel, Switzerland) were eligible for inclusion in the trial. Clusters were randomly assigned to PBO LLINs (PermaNet 3.0 or Olyset Plus) and conventional LLINs (PermaNet 2.0 or Olyset Net) with proportionate randomisation using STATA version 14.2. LLINs were delivered from March 25, 2017, to March 18, 2018. Between April 23, 2019, and Sept 13, 2019, community surveys were conducted in 50 randomly selected households per cluster; ten households per cluster were randomly selected for entomology surveys. Mosquitoes were collected in the morning from indoor surfaces of households using Prokopack aspirators. Due to COVID-19 restrictions, only 90 of the 104 clusters were surveyed at 25 months. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population, determined using the results from the 6-month household survey on the type of LLINs received in each cluster. This trial is registered with ISRCTN, ISRCTN17516395, and is now completed. FINDINGS: In the as-treated analysis, two clusters were excluded (no predominant LLIN received) and four were reassigned; 40 PBO LLIN clusters (30 PermaNet 3.0, ten Olyset Plus) and 48 non-PBO LLIN (36 PermaNet 2.0, 12 Olyset Net) were included. Parasite prevalence was 17·1% (506 of 2958 participants) in the PBO group and 19·8% (701 of 3534) in the non-PBO group (prevalence ratio adjusted for baseline 0·80 [95% CI 0·69-0·93], p=0·0048). Comparing within-treatment group parasite prevalence to baseline, parasite prevalence ratios were lower in the PBO groups at all timepoints, but the difference was greatest at 6 months (PBO LLINs parasite prevalence at baseline 28·8% [1001 of 3472, 95% CI 27·3-30·4] vs at 6 months 12·0% [361 of 3009, 10·9-13·2], prevalence ratio [PR] 0·43 [95% CI 0·36-0·52], p<0·0001; non-PBO LLINs parasite prevalence at baseline 25·4% [1015 of 4004, 24·0-26·7] vs 6 months 14·8% [526 of 3551, 13·7-16·0], PR 0·60 [0·54-0·68], p<0·0001) and 25 months (PBO LLINs parasite prevalence at 25 months 17·1% [506 of 2958, 15·8-18·5], PR 0·63 [95% CI 0·57-0·71], p<0·0001; non-PBO LLINs parasite prevalence at 25 months 19·8% [701 of 3534, 18·5-21·2], PR 0·79 [0·73-0·86], p<0·0001). INTERPRETATION: In Uganda, PBO LLINs outperformed pyrethroid-only LLINs for 25 months. WHO concluded that PBO LLINs are more effective against malaria than non-PBO LLINs when resistance to pyrethroids is high and issued a conditional recommendation suggesting PBO LLINs should be deployed in areas of pyrethroid resistance. FUNDING: The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Identification of a rapidly-spreading triple mutant for high-level metabolic insecticide resistance in Anopheles gambiae provides a real-time molecular diagnostic for antimalarial intervention deployment.

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 resistance-breaking 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 from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) 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 metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

LLIN evaluation in Uganda project (LLINEUP): The fabric integrity, chemical content and bioefficacy of long-lasting insecticidal nets treated with and without piperonyl butoxide across two years of operational use in Uganda.

Long-lasting insecticidal nets (LLINs) supplemented with the synergist piperonyl butoxide have been developed in response to growing pyrethroid resistance; however, their durability in the field remains poorly described. A pragmatic cluster-randomised trial was embedded into Uganda's 2017-2018 LLIN distribution to compare the durability of LLINs with and without PBO. A total of 104 clusters (health sub-districts) were included with each receiving one of four LLIN products, two with pyrethroid + PBO (Olyset Plus and PermaNet 3.0) and two pyrethroid-only (Olyset Net and PermaNet 2.0). Nets were sampled at baseline, 12 and 25 months post-distribution to assess physical condition, chemical content, and bioefficacy. Physical condition was quantified using proportionate Hole Index and chemical content measured using high-performance liquid chromatography. Bioefficacy was assessed with three-minute World Health Organisation (WHO) Cone and Wireball assays using pyrethroid-resistant Anopheles gambiae, with 1-h knockdown and 24-h mortality recorded. There was no difference in physical durability between LLIN products assessed (P = 0.644). The pyrethroid content of all products remained relatively stable across time-points but PBO content declined by 55% (P < 0.001) and 58% (P < 0.001) for Olyset Plus and PermaNet 3.0 respectively. Both PBO LLINs were highly effective against pyrethroid-resistant mosquitoes when new, knocking down all mosquitoes. However, bioefficacy declined over time with Olyset Plus knocking down 45.72% (95% CI: 22.84-68.62%, P = 0.021) and Permanent 3.0 knocking down 78.57% (95% CI: 63.57-93.58%, P < 0.001) after 25 months. 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.

Modelling spatiotemporal trends in the frequency of genetic mutations conferring insecticide target-site resistance in African mosquito malaria vector species.

BACKGROUND: Resistance in malaria vectors to pyrethroids, the most widely used class of insecticides for malaria vector control, threatens the continued efficacy of vector control tools. Target-site resistance is an important genetic resistance mechanism caused by mutations in the voltage-gated sodium channel (Vgsc) gene that encodes the pyrethroid target-site. Understanding the geographic distribution of target-site resistance, and temporal trends across different vector species, can inform strategic deployment of vector control tools. RESULTS: We develop a Bayesian statistical spatiotemporal model to interpret species-specific trends in the frequency of the most common resistance mutations, Vgsc-995S and Vgsc-995F, in three major malaria vector species Anopheles gambiae, An. coluzzii, and An. arabiensis over the period 2005-2017. The models are informed by 2418 observations of the frequency of each mutation in field sampled mosquitoes collected from 27 countries spanning western and eastern regions of Africa. For nine selected countries, we develop annual predictive maps which reveal geographically structured patterns of spread of each mutation at regional and continental scales. The results show associations, as well as stark differences, in spread dynamics of the two mutations across the three vector species. The coverage of ITNs was an influential predictor of Vgsc allele frequencies, with modelled relationships between ITN coverage and allele frequencies varying across species and geographic regions. We found that our mapped Vgsc allele frequencies are a significant partial predictor of phenotypic resistance to the pyrethroid deltamethrin in An. gambiae complex populations. CONCLUSIONS: Our predictive maps show how spatiotemporal trends in insecticide target-site resistance mechanisms in African An. gambiae vary across individual vector species and geographic regions. Molecular surveillance of resistance mechanisms will help to predict resistance phenotypes and track their spread.

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae.

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa. In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

Site-Directed φC31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria.

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the φC31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces φC31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the φC31 integrase. We report two representative results of φC31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. φC31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the φC31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

Effect of long-lasting insecticidal nets with and without piperonyl butoxide on malaria indicators in Uganda (LLINEUP): a pragmatic, cluster-randomised trial embedded in a national LLIN distribution campaign.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are the primary malaria prevention tool, but their effectiveness is threatened by pyrethroid resistance. We embedded a pragmatic cluster-randomised trial into Uganda's national LLIN campaign to compare conventional LLINs with those containing piperonyl butoxide (PBO), a synergist that can partially restore pyrethroid susceptibility in mosquito vectors. METHODS: 104 health sub-districts, from 48 districts in Uganda, were randomly assigned to LLINs with PBO (PermaNet 3.0 and Olyset Plus) and conventional LLINs (PermaNet 2.0 and Olyset Net) by proportionate randomisation using an iterative process. At baseline 6, 12, and 18 months after LLIN distribution, cross-sectional surveys were done in 50 randomly selected households per cluster (5200 per survey); a subset of ten households per cluster (1040 per survey) were randomly selected for entomological surveys. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population at 6, 12, and 18 months. This trial is registered with ISRCTN, ISRCTN17516395. FINDINGS: LLINs were delivered to households from March 25, 2017, to March 18, 2018, 32 clusters were randomly assigned to PermaNet 3.0, 20 to Olyset Plus, 37 to PermaNet 2.0, and 15 to Olyset Net. In the as-treated analysis, three clusters were excluded because no dominant LLIN was received, and four clusters were reassigned, resulting in 49 PBO LLIN clusters (31 received PermaNet 3.0 and 18 received Olyset Plus) and 52 non-PBO LLIN clusters (39 received PermaNet 2.0 and 13 received Olyset Net). At 6 months, parasite prevalence was 11% (386/3614) in the PBO group compared with 15% (556/3844) in the non-PBO group (prevalence ratio [PR] adjusted for baseline values 0·74, 95% CI 0·62-0·87; p=0·0003). Parasite prevalence was similar at month 12 (11% vs 13%; PR 0·73, 95% CI 0·63-0·85; p=0·0001) and month 18 (12% vs 14%; PR 0·84, 95% CI 0·72-0·98; p=0·029). INTERPRETATION: In Uganda, where pyrethroid resistance is high, PBO LLINs reduced parasite prevalence more effectively than did conventional LLINs for up to 18 months. This study provides evidence needed to support WHO's final recommendation on use of PBO LLINs. FUNDING: The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

A high throughput multi-locus insecticide resistance marker panel for tracking resistance emergence and spread in Anopheles gambiae.

The spread of resistance to insecticides in disease-carrying mosquitoes poses a threat to the effectiveness of control programmes, which rely largely on insecticide-based interventions. Monitoring mosquito populations is essential, but obtaining phenotypic measurements of resistance is laborious and error-prone. High-throughput genotyping offers the prospect of quick and repeatable estimates of resistance, while also allowing resistance markers to be tracked and studied. To demonstrate the potential of highly-mulitplexed genotypic screening for measuring resistance-association of mutations and tracking their spread, we developed a panel of 28 known or putative resistance markers in the major malaria vector Anopheles gambiae, which we used to screen mosquitoes from a wide swathe of Sub-Saharan Africa (Burkina Faso, Ghana, Democratic Republic of Congo (DRC) and Kenya). We found resistance association in four markers, including a novel mutation in the detoxification gene Gste2 (Gste2-119V). We also identified a duplication in Gste2 combining a resistance-associated mutation with its wild-type counterpart, potentially alleviating the costs of resistance. Finally, we describe the distribution of the multiple origins of kdr resistance, finding unprecedented diversity in the DRC. This panel represents the first step towards a quantitative genotypic model of insecticide resistance that can be used to predict resistance status in An. gambiae.

LLIN Evaluation in Uganda Project (LLINEUP): factors associated with childhood parasitaemia and anaemia 3 years after a national long-lasting insecticidal net distribution campaign: a cross-sectional survey.

BACKGROUND: Recent reductions in malaria burden have been attributed largely to long-lasting insecticidal nets (LLINs). In March-June 2017, approximately 3 years after a national LLIN distribution campaign, a cross-sectional community survey was conducted to investigate factors associated with malaria parasitaemia and anaemia, in advance of Uganda's 2017-2018 LLIN campaign. METHODS: Households from 104 clusters in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Eligible children aged 2-10 years had blood obtained for a thick blood smear and those aged 2-4 years had haemoglobin measured. Associations between outcomes and variables of interest were assessed using log-binomial regression with generalized estimating equations to adjust for household clustering. RESULTS: In total, 5196 households, 8834 children with blood smear results, and 3753 with haemoglobin results were included. Only 16% of children lived in households with adequate LLIN coverage. Overall, parasite prevalence was 26.0%, ranging from 8.0% in the South West to 53.1% in East Central. Limiting data to children 2-4 years of age, parasite prevalence was 21.4%, up from 16.9% in 2014-2015 following the national LLIN campaign. In a multivariate analysis, factors associated with parasitaemia included region (East-Central vs South-Western; adjusted prevalence ratio [aPR] 6.45, 95% CI 5.55-7.50; p < 0.001), older age (8-10 vs 2-3 years; aPR 1.57, 95% CI 1.43-1.72; p < 0.001), living in a poorer household (poorest vs least poor tercile; aPR 2.32, 95% CI 2.05-2.63; p < 0.001), one constructed of traditional materials (aPR 1.13, 95% CI 1.03-1.24; p = 0.008), or without adequate LLIN coverage (aPR 1.30, 95% CI 1.14-1.48; p < 0.001). Overall, the prevalence of anaemia (haemoglobin < 10 g/dL) was 15.1% and varied geographically. In a multivariate analysis, factors associated with anaemia included region, younger age, living in a traditional house, and parasitaemia, which was the strongest predictor (aPR 2.50, 95% CI 2.12-2.95; p < 0.001). CONCLUSIONS: Three years after a national LLIN campaign, LLIN coverage was low and parasite prevalence had increased. Parasite prevalence varied widely across Uganda; older children, those living in poorer households, and those with inadequate LLIN coverage, were at highest risk of parasitaemia. LLINs may need to be distributed more frequently through mass campaigns or continuously through sustainable mechanisms. Targeting interventions to geographic areas and populations at highest risk should also be considered.

LLIN Evaluation in Uganda Project (LLINEUP) - Impact of long-lasting insecticidal nets with, and without, piperonyl butoxide on malaria indicators in Uganda: study protocol for a cluster-randomised trial.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention, but their effectiveness is threatened by resistance to pyrethroid insecticides. Some new LLINs combine pyrethroids with piperonyl butoxide (PBO), a synergist that can overcome P450-based metabolic resistance to pyrethroids in mosquitoes. In 2017-2018, the Ugandan Ministry of Health distributed LLINs with and without PBO through a national mass-distribution campaign, providing a unique opportunity to rigorously evaluate PBO LLINs across different epidemiological settings. METHODS/DESIGN: Together with the Ministry of Health, we embedded a cluster-randomised trial to evaluate the impact of LLINs delivered in the 2017-2018 national campaign. A total of 104 clusters (health sub-districts) in Eastern and Western Uganda were involved, covering 48 of 121 (40%) districts. Using adaptive randomisation driven by the number of LLINs available, clusters were assigned to receive one of four types of LLINs, including two brands with PBO: 1) PermaNet 3.0 (n = 32) and 2) Olyset Plus (n = 20); and two without PBO: 3) PermaNet 2.0 (n = 37) and 4) Olyset Net (n = 15). We are conducting cross-sectional community surveys in 50 randomly selected households per cluster (5200 households per survey) and entomological surveillance for insecticide resistance in up to 10 randomly selected households enrolled in the community surveys per cluster (1040 households per survey) at baseline and 6, 12, and 18 months after LLIN distribution. Net durability and bio-efficacy will be assessed in 400 nets withdrawn from households with replacement at 12 months. The primary trial outcome is parasite prevalence as measured by microscopy in children aged 2-10 years in the follow-up surveys. DISCUSSION: PBO LLINs are a promising new tool to reduce the impact of pyrethroid resistance on malaria control. The World Health Organization has issued a preliminary endorsement of PBO LLINs, but additional epidemiological evidence of the effect of PBO LLINs is urgently needed. The results of this innovative, large-scale trial embedded within a routine national distribution campaign will make an important contribution to the malaria control policy in Uganda and throughout Africa, where pyrethroid resistance in malaria vectors has increased dramatically. This model of evaluation could be a paradigm for future assessment of malaria control interventions. TRIAL REGISTRATION: ISRCTN, ISRCTN17516395 . Registered on 14 February 2017. WORLD HEALTH ORGANIZATION TRIAL REGISTRATION DATA SET: See Additional file 1.

LLIN Evaluation in Uganda Project (LLINEUP): a cross-sectional survey of species diversity and insecticide resistance in 48 districts of Uganda.

BACKGROUND: Long-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. RESULTS: Prior 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. CONCLUSIONS: No 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/ISRCTN17516395.

Insecticide resistance in Anopheles gambiae from the northern Democratic Republic of Congo, with extreme knockdown resistance (kdr) mutation frequencies revealed by a new diagnostic assay.

BACKGROUND: Mutations 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. METHODS: Samples 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. RESULTS: Anopheles 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. CONCLUSION: This 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.

LLIN Evaluation in Uganda Project (LLINEUP): factors associated with ownership and use of long-lasting insecticidal nets in Uganda: a cross-sectional survey of 48 districts.

BACKGROUND: Long-lasting insecticidal nets (LLINs) are a key malaria control intervention. To investigate factors associated with ownership and use of LLINs in Uganda, a cross-sectional community survey was conducted in March-June 2017, approximately 3 years after a national Universal Coverage Campaign (UCC). METHODS: Households from 104 clusters (health sub-districts) in 48 districts were randomly selected using two-staged cluster sampling; 50 households were enrolled per cluster. Outcomes were household ownership of LLINs (at least one LLIN), adequate LLIN coverage (at least one LLIN per 2 residents), and use of LLINs (resident slept under a LLIN the previous night). Associations between variables of interest and outcomes were made using multivariate logistic regression. RESULTS: In total, 5196 households, with 29,627 residents and 6980 bed-nets, were included in the analysis. Overall, 65.0% of households owned at least one LLIN (down from 94% in 2014). In the adjusted analysis, factors most strongly associated with LLIN ownership were living in a wealthier household (highest tercile vs lowest; adjusted odds ratio [aOR] 1.94, 95% CI 1.66-2.28, p < 0.001) and time since the last UCC (29-37 vs 42-53 months; aOR 1.91, 95% CI 1.60-2.28, p < 0.001). Only 17.9% of households had adequate LLIN coverage (down from 65% in 2014). Factors most strongly associated with adequate coverage were fewer residents (2-4 vs ≥ 7; aOR 6.52, 95% CI 5.13-8.29, p < 0.001), living in a wealthier household (highest tercile vs lowest; aOR: 2,32, 95% CI 1.88-2.85, p < 0.001) and time since the last UCC (29-37 vs 42-53 months; aOR 2.13, 95% CI 1.61-2.81, p < 0.001). Only 39.5% of residents used a LLIN the previous night. Age was strongly associated with LLIN use, as were household wealth and time since the last UCC. Children < 5 years (44.7%) and residents > 15 years (44.1%) were more likely to use nets than children aged 5-15 years (30.7%; < 5 years: aOR 1.71, 95% CI 1.62-1.81, p < 0.001; > 15 years: aOR 1.37, 95% CI 1.29-1.45, p < 0.001). CONCLUSIONS: Long-lasting insecticidal net ownership and coverage have reduced markedly in Uganda since the last net distribution campaign in 2013/14. Houses with many residents, poorer households, and school-aged children should be targeted to improve LLIN coverage and use. Trial registration This study is registered with ISRCTN (17516395).

Multi-tissue GAL4-mediated gene expression in all Anopheles gambiae life stages using an endogenous polyubiquitin promoter.

The ability to manipulate the Anopheles gambiae genome and alter gene expression effectively and reproducibly is a prerequisite for functional genetic analysis and for the development of novel control strategies in this important disease vector. However, in vivo transgenic analysis in mosquitoes is limited by the lack of promoters active ubiquitously. To address this, we used the GAL4/UAS system to investigate the promoter of the An. gambiae Polyubiquitin-c (PUBc) gene and demonstrated its ability to drive expression in mosquito cell culture before incorporation into An. gambiae transgenic driver lines. To generate such lines, piggyBac-mediated insertion was used to identify genomic regions able to sustain widespread expression and to create φC31 docking lines at these permissive sites. Patterns of expression induced by PUBc-GAL4 drivers carrying single intergenic insertions were assessed by crossing with a novel responder UAS-mCD8:mCherry line that was created by φC31-mediated integration. Amongst the drivers created at single, unique chromosomal integration loci, two were isolated that induced differential expression levels in a similar multiple-tissue spatial pattern throughout the mosquito life cycle. This work expands the tools available for An. gambiae functional analysis by providing a novel promoter for investigating phenotypes resulting from widespread multi-tissue expression, as well as identifying and tagging genomic sites that sustain broad transcriptional activity.

Insecticide-induced leg loss does not eliminate biting and reproduction in Anopheles gambiae mosquitoes.

Recent successes in malaria control have been largely attributable to the deployment of insecticide-based vector control tools such as bed nets and indoor residual spraying. Pyrethroid-treated bed nets are acutely neurotoxic to mosquitoes, inducing symptoms such as loss of coordination, paralysis, and violent spasms. One result of pyrethroid exposure often seen in laboratory tests is mosquito leg loss, a condition that has thus far been assumed to equate to mortality, as females are not expected to blood feed. However, whilst limb loss is unlikely to be adaptive, females with missing limbs may play a role in the propagation of both their species and pathogens. To test the hypothesis that leg loss inhibits mosquitoes from biting and reproducing, mosquitoes with one, two, or six legs were evaluated for their success in feeding upon a human. These experiments demonstrated that insecticide-induced leg loss had no significant effect upon blood feeding or egg laying success. We conclude that studies of pyrethroid efficacy should not discount mosquitoes that survive insecticide exposure with fewer than six legs, as they may still be capable of biting humans, reproducing, and contributing to malaria transmission.