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Abstract Robust diagnostic tools and surveillance are crucial for malaria control and elimination efforts. Malaria caused by neglected Plasmodium parasites is often underestimated due to the lack of rapid diagnostic tools that can accurately detect these species. While nucleic-acid amplification technologies stand out as the most sensitive methods for detecting and confirming Plasmodium species, their implementation in resource-constrained settings poses significant challenges. Here, we present a Pan Plasmodium recombinase polymerase amplification lateral flow (RPA–LF) assay, capable of detecting all six human infecting Plasmodium species in low resource settings. The Pan Plasmodium RPA-LF assay successfully detected low density clinical infections with a preliminary limit of detection between 10–100 fg/µl for P. falciparum. When combined with crude nucleic acid extraction, the assay can serve as a point-of-need tool for molecular xenomonitoring. This utility was demonstrated by screening laboratory-reared Anopheles stephensi mosquitoes fed with Plasmodium-infected blood, as well as field samples of An. funestus s.l. and An. gambiae s.l. collected from central Africa. Overall, our proof-of-concept Pan Plasmodium diagnostic tool has the potential to be applied for clinical and xenomonitoring field surveillance, and after further evaluation, could become an essential tool to assist malaria control and elimination.

Abstract The present study aimed to assess mosquito species diversity, distribution, and ecological preferences in the Covè, Ouinhi, and Zangnanado communes, Southern Benin. Such information is critical to understand mosquito bio-ecology and to focus control efforts in high-risk areas for vector-borne diseases. Mosquito collections occurred quarterly in 60 clusters between June 2020 and April 2021, using human landing catches. In addition to the seasonal mosquito abundance, Shannon's diversity, Simpson, and Pielou's equitability indices were also evaluated to assess mosquito diversity. Ecological niche models were developed with MaxEnt using environmental variables to assess species distribution. Overall, mosquito density was higher in the wet season than in the dry season in all communes. A significantly higher Shannon's diversity index was also observed in the wet season than in the dry seasons in all communes (p

Abstract Aedes aegypti is vector of many arboviruses including Zika, dengue, yellow fever, West Nile, and Chikungunya. Its control efforts are hampered by widespread insecticide resistance reported in the Americas and Asia, while data from Africa is more limited. Here we use publicly available 729 Ae. aegypti whole-genome sequencing samples from 15 countries, including nine in Africa, to investigate the genetic diversity in four insecticide resistance linked genes: ace-1, GSTe2, rdl and vgsc. Apart from vgsc, the other genes have been less investigated in Ae. aegypti, and almost no genetic diversity information is available. Among the four genes, we identified 1,829 genetic variants including 474 non-synonymous substitutions, some of which have been previously documented, as well as putative copy number variations in GSTe2 and vgsc. Global insecticide resistance phenotypic data demonstrated variable resistance in geographic areas with resistant genotypes. Overall, our work provides the first global catalogue and geographic distribution of known and new amino-acid mutations and duplications that can be used to guide the identification of resistance drivers in Ae. aegypti and thereby support monitoring efforts and strategies for vector control.

Abstract The present cluster-randomised control trial aims to assess the entomological efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs compared to the standard pyrethroid-only LLINs, in their third year of community usage. Adult mosquito collections were performed every 3 months, in 4 randomly selected houses in each of the 60 trial clusters, using human landing catches. Adult mosquitoes were morphologically identified and Anopheles vectors were molecularly speciated and screened for the presence of the L1014F kdr mutation using PCR. Plasmodium falciparum sporozoite infection was assessed using ELISA. A subset of An. gambiae s.l. was also dissected to examine parity and fertility rates across study arms. There was no evidence of a significant reduction in indoor vector density and entomological inoculation rate by the pyrethroid-pyriproxyfen [DR 0.94 (95% CI 0.46–1.88), p = 0.8527; and RR 1.10 (95% CI 0.44–2.72), p = 0.8380], and pyrethroid-chlorfenapyr [DR 0.74 (95% CI 0.37–1.48), p = 0.3946; and RR 1.00 (95% CI 0.40–2.50), p = 0.9957] LLINs, respectively. The same trend was observed outdoors. Frequencies of the L1014F kdr mutation, as well as parous and fertility rates, were similar between study arms. In the third year after net distribution, entomological indicators show that the two dual active-ingredients nets performed similarly to the standard pyrethroid-only LLIN. To maintain malaria gains, it is crucial that net distribution cycles fit with their operational lifespan.

Abstract Background Recently, bacterial endosymbiont, including Wolbachia and Microsporidia were found to limit the infection of Anopheles mosquitoes with Plasmodium falciparum. This study aimed to investigate the natural presence of key transmission-blocking endosymbionts in Anopheles gambiae and Anopheles coluzzii in Southern Benin. Methods The present study was conducted in seven communes (Cotonou, Porto-Novo, Aguégués, Ifangni, Pobè Athiémé, and Grand-Popo) of Southern Benin. Anopheles were collected using indoor/outdoor Human Landing Catches (HLCs) and Pyrethrum Spray Catches (PSCs). Following morphological identification, PCR was used to identify An. gambiae sensu lato (s.l.) to species level and to screen for the presence of both Wolbachia and Microsporidia. Plasmodium falciparum sporozoite infection was also assessed using ELISA. Results Overall, species composition in An. gambiae s.l. was 53.7% An. coluzzii, while the remainder was An. gambiae sensu stricto (s.s.). Combined data of the two sampling techniques revealed a mean infection prevalence with Wolbachia of 5.1% (95% CI 0.90–18.6) and 1.3% (95% CI 0.07–7.8) in An. gambiae s.s. and An. coluzzii, respectively. The mean infection prevalence with Microsporidia was 41.0% (95% CI 25.9–57.8) for An. gambiae s.s. and 57.0% (95% CI 45.4–67.9) for An. coluzzii. Wolbachia was only observed in Ifangni, Pobè, and Cotonou, while Microsporidia was detected in all study communes. Aggregated data for HLCs and PSCs showed a sporozoite rate (SR) of 0.80% (95% CI 0.09–2.87) and 0.69% (95% CI 0.09–2.87) for An. gambiae and An. coluzzii, respectively, with a mean of 0.74% (95% CI 0.20–1.90). Of the four individual mosquitoes which harboured P. falciparum, none were also infected with Wolbachia and one contained Microsporidia. Conclusions The present study is the first report of natural infections of field-collected An. gambiae s.l. populations from Benin with Wolbachia and Microsporidia. Sustained efforts should be made to widen the spectrum of bacteria identified in mosquitoes, with the potential to develop endosymbiont-based control tools; such interventions could be the game-changer in the control of malaria and arboviral disease transmission.

Abstract Background The massive scale-up of long-lasting insecticidal nets (LLIN) has led to a major reduction in malaria burden in many sub-Saharan African (SSA) countries. The World Health Organization (WHO) has recently issued a strong recommendation for the use of chlorfenapyr-pyrethroid LLINs compared to standard pyrethroid-only LLINs in areas of high insecticide resistance intensity. However, there is still a lack of conclusive evidence on the efficacy of piperonyl butoxide-pyrethroid (PBO-py) LLINs, especially in West Africa, where vector composition and resistance mechanisms may be different from vectors in East Africa. Methods This is a three-arm, superiority, triple-blinded, cluster randomised trial, with village as the unit of randomisation. This study conducted in Côte d’Ivoire will evaluate the efficacy on epidemiological and entomological outcomes of (1) the control arm: MAGNet® LN, which contains the pyrethroid, alpha-cypermethrin, (2) VEERALIN® LN, a net combining the synergist PBO and alpha-cypermethrin, and (3) Interceptor® G2 LN, which incorporates chlorfenapyr and alpha-cypermethrin, two adulticides with different mechanisms of action. A total of 33 villages with an average of 200 households per village will be identified, mapped, and randomised in a ratio of 1:1:1. Nets will be distributed at a central point following national guidelines with 1 net for every 2 people. The primary outcome of the trial will be incidence of malaria cases (confirmed by rapid diagnostic test (RDT)) in a cohort of 50 children aged 6 months to 10 years in each cluster, followed for 12 months (active case detection). Secondary outcomes are cross-sectional community prevalence of malaria infection (confirmed by RDT) in the study population at 6 and 12 months post-intervention (50 randomly selected persons per cluster), vector density, entomological inoculation rate (EIR), and phenotypic and genotypic insecticide resistance at baseline and 12 months post-intervention in 3 sentinel villages in each treatment arm. Discussion In addition to generating further evidence for next-generation LLINs, this study will also provide the first evidence for pyrethroid-PBO nets in a West African setting. This could further inform WHO recommendations on the pragmatic use of pyrethroid-PBO nets. Trial registration ClinicalTrials.gov NCT05796193. Registered on April 3, 2023.

Abstract Background Long-lasting insecticidal nets (LLINs) may have different impacts on distinct mosquito vector species. We assessed the efficacy of pyrethroid-pyriproxyfen and pyrethroid-chlorfenapyr LLINs on the density of Anopheles gambiae s.s. and An. coluzzii compared to pyrethroid-only nets in a three-arm cluster randomised control trial in Benin. Methods Indoor and outdoor collections of adult mosquitoes took place in 60 clusters using human landing catches at baseline and every 3 months for 2 years. After morphological identification, around 15% of randomly selected samples of An. gambiae s.l. were dissected to determine parity, species (using PCR). Results Overall, a total of 46,613 mosquito specimens were collected at baseline and 259,250 in the eight quarterly collections post-net distribution. Post-net distribution, approximately 70% of the specimens of An. gambiae s.l. speciated were An. coluzzii, while the rest were mostly composed of An. gambiae s.s. with a small proportion (

Abstract Vector control strategies have been successful in reducing the number of malaria cases and deaths globally, but the spread of insecticide resistance represents a significant threat to disease control. Insecticide resistance has been reported across Anopheles (An.) vector populations, including species within the An. funestus group. These mosquitoes are responsible for intense malaria transmission across sub-Saharan Africa, including in the Democratic Republic of the Congo (DRC), a country contributing > 12% of global malaria infections and mortality events. To support the continuous efficacy of vector control strategies, it is essential to monitor insecticide resistance using molecular surveillance tools. In this study, we developed an amplicon sequencing (“Amp-seq”) approach targeting An. funestus, and using multiplex PCR, dual index barcoding, and next-generation sequencing for high throughput and low-cost applications. Using our Amp-seq approach, we screened 80 An. funestus field isolates from the DRC across a panel of nine genes with mutations linked to insecticide resistance (ace-1, CYP6P4, CYP6P9a, GSTe2, vgsc, and rdl) and mosquito speciation (cox-1, mtND5, and ITS2). Amongst the 18 non-synonymous mutations detected, was N485I, in the ace-1 gene associated with carbamate resistance. Overall, our panel represents an extendable and much-needed method for the molecular surveillance of insecticide resistance in An. funestus populations.

Abstract Background After decades of success in reducing malaria through the scale-up of pyrethroid long-lasting insecticidal nets (LLINs), the decline in the malaria burden has stalled, coinciding with the rapid spread of pyrethroid resistance. In a previously reported study, nets treated with a pyrethroid and a synergist, piperonyl butoxide (PBO), demonstrated superior efficacy compared to standard pyrethroid LLINs (std-LLINs) against malaria. Evidence was used to support the public health recommendation of PBO-Pyrethroid-LLIN by the World Health Organization in 2018. This study looks at the third year of rollout of these nets in Muleba district, Tanzania to inform whether policy guidelines need to be updated. Methods A four-group cluster randomized trial (CRT) using a two-by-two factorial design was carried out between January 2014 and December 2017. A total of 48 clusters, were randomized in a 1:1:1:1 ratio to the following treatment groups, each intervention being provided once in 2015: 1/std-LLIN; 2/PBO-pyrethroid LLIN; 3/std-LLIN + Indoor Residual Spraying (IRS) and 4/PBO-Pyrethroid-LLIN + IRS. During the third year follow-up, malaria infection prevalence in 80 children per cluster, aged 6 months to 14 years, was measured at 28- and 33-months post-intervention and analysed as intention-to-treat (ITT) and per protocol (PP). Mosquito collections were performed monthly in all clusters, using CDC light traps in 7 randomly selected houses per cluster. Results At 28 and 33 months, study net usage among household participants was only 47% and 31%, respectively. In ITT analysis, after 28 months malaria infection prevalence among 7471 children was 80.9% in the two std-LLIN groups compared to 69.3% in the two PBO-Pyrethroid-LLIN (Odds Ratio: 0.45, 95% Confidence Interval: 0.21–0.95, p-value: 0.0364). After 33 months the effect was weaker in the ITT analysis (prevalence 59.6% versus 49.9%, OR: 0.60, 95%CI:0.32–1.13, p-value: 0.1131) but still evident in the PP analysis (57.2% versus 44.2%, OR: 0.34, 95%CI: 0.16–0.71, p-value: 0.0051). Mean number of Anopheles per night collected per house was similar between PBO-Pyrethroid-LLIN groups (5.48) and std-LLIN groups (5.24) during the third year. Conclusions Despite low usage of PBO- Pyrethroid LLIN, a small impact of those nets on malaria infection prevalence was still observed in the 3rd year with the most protection offered to children still using them. To maximize impact, it is essential that net re-distribution cycles are aligned with this LLIN lifespan to maintain maximum coverage. Trial registration: The trial was registered with ClinicalTrials.gov (registration number NCT02288637).

Abstract Selection of mosquito collection methods is of crucial importance to evaluate the impact of vector control tools on entomological outcomes. During a cluster randomised control trial evaluating the relative efficacy of two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) compared to pyrethroid-only LLINs, we assessed the performance of different mosquito collection methods: Human landing catches (HLC), Centers for Disease Control and Prevention (CDC) light traps, and pyrethrum spray catches (PSC). Anopheles mosquitoes were collected using three collection methods in 4 houses, in each of the 60 trial clusters at baseline and every quarter for 24 months using PSCs and HLCs, while CDC light traps were performed during two quarters only. Mean density of vectors collected per method per night was the highest with HLCs (15.9), followed by CDC light traps (6.8); with PSCs (1.1) collecting 10 times less mosquitoes than HLCs. All three collection methods collected fewer mosquitoes in the Interceptor G2® dual a.i. arm, compared to the other trial arms, although only HLCs and PSCs demonstrated strong evidence of this due to a greater number of collection rounds undertaken, than CDC light traps. The broadly similar results regarding the differential impact of the two dual a.i. LLINs showed by the three collection methods suggest that the more ethically acceptable, cheaper, and logistically simpler methods such as CDC light traps could be prioritised for use in large community trials for measuring the efficacy of vector control tools.

Epidemics of arboviruses in general, and dengue fever in particular, are an increasing threat in areas where Aedes (Ae.) aegypti is present. The effectiveness of chemical control of Ae. aegypti is jeopardized by the increasing frequency of insecticide resistance. The aim of this study was to determine the susceptibility status of Ae. aegypti to public health insecticides and assess the underlying mechanisms driving insecticide resistance. Ae. aegypti eggs were collected in two study sites in the vicinity of houses for two weeks using gravid Aedes traps (GATs). After rearing the mosquitoes to adulthood, female Ae. aegypti were exposed to diagnostic doses of permethrin, deltamethrin and bendiocarb, using Centers for Disease Control and Prevention (CDC) bottle bioassays. Unexposed, un-engorged female Ae. aegypti were tested individually for mixed-function oxidase (MFO), glutathione-S-transferase (GST) and α and β esterase activities. Finally, allele-specific PCR (AS-PCR) was used to detect possible kdr mutations (F1534C, S989P, and V1016G) in the voltage-gated sodium channel gene in insecticide-exposed Ae. aegypti. Most traps were oviposition positive; 93.2% and 97% of traps contained Ae. aegypti eggs in the 10ème arrondissement of Cotonou and in Godomey-Togoudo, respectively. Insecticide bioassays detected resistance to permethrin and deltamethrin in both study sites and complete susceptibility to bendiocarb. By comparison to the insecticide-susceptible Rockefeller strain, field Ae. aegypti populations had significantly higher levels of GSTs and significantly lower levels of α and β esterases; there was no significant difference between levels of MFOs. AS-PCR genotyping revealed the possible presence of 3 kdr mutations (F1534C, S989P, and V1016G) at high frequencies; 80.9% (228/282) of the Ae. aegypti tested had at least 1 mutation, while the simultaneous presence of all 3 kdr mutations was identified in 13 resistant individuals. Study findings demonstrated phenotypic pyrethroid resistance, the over-expression of key detoxification enzymes, and the possible presence of several kdr mutations in Ae. aegypti populations, emphasizing the urgent need to implement vector control strategies targeting arbovirus vector species in Benin.

Abstract Since its first detection in 2012 in Djibouti, Anopheles stephensi has invaded and established in the Horn of Africa, and more recently Nigeria. The expansion of this vector poses a significant threat to malaria control and elimination efforts. Integrated vector management is the primary strategy used to interrupt disease transmission; however, growing insecticide resistance is threatening to reverse gains in global malaria control. We present a next-generation amplicon-sequencing approach, for high-throughput monitoring of insecticide resistance genes (ace1, GSTe2, vgsc and rdl), species identification and characterization of genetic diversity (its2 and cox1) in An. stephensi. Ninety-five An. stephensi mosquitoes, collected in Ethiopia, were screened, identifying 104 SNPs, including the knock-down mutation L958F (L1014F in Musca domestica), and for the first time in this vector species, the A296S substitution (A301S in Drosophila melanogaster) in the rdl locus. Two other amino acid substitutions (ace1-N177D, GSTe2-V189L) were also identified but have not been previously implicated in insecticide resistance. Genetic diversity in the mitochondrial cox1 gene revealed shared haplotypes between Ethiopian An. stephensi with samples from Pakistan, Sudan, and Djibouti. Overall, we present a reliable, cost-effective strategy using amplicon-sequencing to monitor known insecticide resistance mutations, with the potential to identify new genetic variants, to assist in the high-throughput surveillance of insecticide resistance in An. stephensi populations.

Abstract The invasion and establishment of An. stephensi mosquitoes in the Horn of Africa represents a significant regional threat, which may jeopardise malaria control, particularly in urban areas which were formally free from disease transmission. Novel vector surveillance methods are urgently needed, both agnostic to mosquito larval morphology, and simple to implement at the sampling stage. Using new multiplex TaqMan assays, specifically targeting An. stephensi and Ae. aegypti, we validated the use of environmental DNA (eDNA) for simultaneous vector detection in shared artificial breeding sites. Study findings demonstrated that An. stephensi and Ae. aegypti eDNA deposited by as few as one second instar larva in 1L of water was detectable. Characterization of molecular insecticide resistance mechanisms, using novel amplicon-sequencing panels for both vector species, was possible from eDNA shed by as few as 16–32 s instar larvae in 50 ml of water. An. stephensi eDNA, derived from emergent pupae for 24 h, was remarkably stable, and still detectable ~ 2 weeks later. eDNA surveillance has the potential to be implemented in local endemic communities and at points of country entry, to monitor the spread of invasive vector species. Further studies are required to validate the feasibility of this technique under field conditions.

Pyrethroid-treated long-lasting insecticidal nets (LLINs) have been the main contributor to the reduction in malaria in the past two decades in sub-Saharan Africa. The development of pyrethroid insecticide resistance threatens the future of LLINs, especially when nets become holed and pyrethroid decays. In this study, three new classes of dual-active ingredient (AI) LLINs were evaluated for their physical durability: (1) Royal Guard, combining pyriproxyfen, which disrupts female fertility, and a pyrethroid, alpha-cypermethrin; (2) Interceptor G2, which combines the pyrrole chlorfenapyr and a pyrethroid (alpha-cypermethrin); (3) Olyset Plus, which incorporates the pyrethroid permethrin and the synergist piperonyl butoxide, to enhance the pyrethroid potency; and Interceptor, a reference net that contains alpha-cypermethrin as the sole active ingredient. About 40,000 nets of each type were distributed in February 2019 to different villages in Misungwi. A total of 3072 LLINs were followed up every 6–12 months up to 36 months to assess survivorship and fabric integrity. The median functional survival was less than three years with Interceptor, Interceptor G2, and Royal Guard showing 1.9 years each and Olyset Plus showing 0.9 years. After 36 months, 90% of Olyset Plus and Royal Guard and 87% of Interceptor G2 were no longer in use (discarded) due to wear and tear, compared to 79% for Interceptor. All dual-AI LLINs exhibited poor textile durability, with Olyset Plus being the worst.

Abstract Novel insecticides are urgently needed to control insecticide-resistant populations of Anopheles malaria vectors. Broflanilide acts as a non-competitive antagonist of the gamma-aminobutyric acid receptor and has shown prolonged effectiveness as an indoor residual spraying product (VECTRON T500) in experimental hut trials against pyrethroid-resistant vector populations. This multi-centre study expanded upon initial discriminating concentration testing of broflanilide, using six Anopheles insectary colonies (An. gambiae Kisumu KCMUCo, An. gambiae Kisumu NIMR, An. arabiensis KGB, An. arabiensis SENN, An. coluzzii N’Gousso and An. stephensi SK), representing major malaria vector species, to facilitate prospective susceptibility monitoring of this new insecticide; and investigated the potential for cross-resistance to broflanilide via the A296S mutation associated with dieldrin resistance (rdl). Across all vector species tested, the discriminating concentration for broflanilide ranged between LC99 × 2 = 1.126–54.00 μg/ml or LC95 × 3 = 0.7437–17.82 μg/ml. Lower concentrations of broflanilide were required to induce complete mortality of An. arabiensis SENN (dieldrin-resistant), compared to its susceptible counterpart, An. arabiensis KGB, and there was no association between the presence of the rdl mechanism of resistance and survival in broflanilide bioassays, demonstrating a lack of cross-resistance to broflanilide. Study findings provide a benchmark for broflanilide susceptibility monitoring as part of ongoing VECTRON T500 community trials in Tanzania and Benin.

Abstract Surveillance of malaria vector species and the monitoring of insecticide resistance are essential to inform malaria control strategies and support the reduction of infections and disease. Genetic barcoding of mosquitoes is a useful tool to assist the high-throughput surveillance of insecticide resistance, discriminate between sibling species and to detect the presence of Plasmodium infections. In this study, we combined multiplex PCR, custom designed dual indexing, and Illumina next generation sequencing for high throughput single nucleotide polymorphism (SNP)-profiling of four species from the Anopheles (An.) gambiae complex (An. gambiae sensu stricto, An. coluzzii, An. arabiensis and An. melas). By amplifying and sequencing only 14 genetic fragments (500 bp each), we were able to simultaneously detect Plasmodium infection; insecticide resistance-conferring SNPs in ace1, gste2, vgsc and rdl genes; the partial sequences of nuclear ribosomal internal transcribed spacers (ITS1 and ITS2) and intergenic spacers (IGS), Short INterspersed Elements (SINE), as well as mitochondrial genes (cox1 and nd4) for species identification and genetic diversity. Using this amplicon sequencing approach with the four selected An. gambiae complex species, we identified a total of 15 non-synonymous mutations in the insecticide target genes, including previously described mutations associated with resistance and two new mutations (F1525L in vgsc and D148E in gste2). Overall, we present a reliable and cost-effective high-throughput panel for surveillance of An. gambiae complex mosquitoes in malaria endemic regions.

Abstract Chagas disease vector control relies on prompt, accurate identification of houses infested with triatomine bugs for targeted insecticide spraying. However, most current detection methods are laborious, lack standardization, have substantial operational costs and limited sensitivity, especially when triatomine bug densities are low or highly focal. We evaluated the use of FTA cards or cotton-tipped swabs to develop a low-technology, non-invasive method of detecting environmental DNA (eDNA) from both triatomine bugs and Trypanosoma cruzi for use in household surveillance in eastern Colombia, an endemic region for Chagas disease. Study findings demonstrated that Rhodnius prolixus eDNA, collected on FTA cards, can be detected at temperatures between 21 and 32 °C, when deposited by individual, recently blood-fed nymphs. Additionally, cotton-tipped swabs are a feasible tool for field sampling of both T. cruzi and R. prolixus eDNA in infested households and may be preferable due to their lower cost. eDNA detection should not yet replace current surveillance tools, but instead be evaluated in parallel as a more sensitive, higher-throughput, lower cost alternative. eDNA collection requires virtually no skills or resources in situ and therefore has the potential to be implemented in endemic communities as part of citizen science initiatives to control Chagas disease transmission.

Abstract Background Despite the low to moderate intensity of malaria transmission present in Ethiopia, malaria is still a leading public health problem. Current vector control interventions, principally long-lasting insecticidal nets and indoor residual spraying, when deployed alone or in combination, are insufficient to control the dominant vector species due to their exophagic and exophilic tendencies. Zooprophylaxis presents a potential supplementary vector control method for malaria; however, supporting evidence for its efficacy has been mixed. Methods To identify risk factors of malaria and to estimate the association between cattle and Anopheles vector abundance as well as malaria risk, a cross-sectional study was conducted in a village near Arba Minch, Ethiopia. Epidemiological surveys (households = 95, individuals = 463), mosquito collections using CDC light traps and a census of cattle and human populations were conducted. To capture environmental conditions, land cover and water bodies were mapped using satellite imagery. Risk factor analyses were performed through logistic, Poisson, negative binomial, and spatial weighted regression models. Results The only risk factor associated with self-reported malaria illness at an individual level was being a child aged 5 or under, where they had three times higher odds than adults. At the household level, variables associated with malaria vector abundance, especially those indoors, included socioeconomic status, the proportion of children in a household and cattle population density. Conclusions Study results are limited by the low abundance of malaria vectors found and use of self-reported malaria incidence. Environmental factors together with a household’s socioeconomic status and host availability played important roles in the risk of malaria infection in southwest Ethiopia. Cattle abundance in the form of higher cattle to human ratios may act as a protective factor against mosquito infestation and malaria risk. Humans should remain indoors to maximize potential protection against vectors and cattle kept outside of homes.

Abstract Background Progress achieved by long-lasting insecticidal nets (LLINs) against malaria is threatened by widespread selection of pyrethroid resistance among vector populations. LLINs with non-pyrethroid insecticides are urgently needed. This study aims to assess the insecticide and textile durability of three classes of dual-active ingredient (A.I.) LLINs using techniques derived from established WHO LLIN testing methods to set new standards of evaluation. Methods A WHO Phase 3 active ingredients and textile durability study will be carried out within a cluster randomized controlled trial in 40 clusters in Misungwi district, Tanzania. The following treatments will be evaluated: (1) Interceptor®G2 combining chlorfenapyr and the pyrethroid alpha-cypermethrin, (2) Royal Guard® treated with pyriproxyfen and alpha-cypermethrin, (3) Olyset™ Plus which incorporates a synergist piperonyl butoxide and the pyrethroid permethrin, and (4) a reference standard alpha-cypermethrin only LLIN (Interceptor®). 750 nets will be followed in 5 clusters per intervention arm at 6, 12, 24 and 36 months post distribution for survivorship and hole index assessment. A second cohort of 1950 nets per net type will be identified in 10 clusters, of which 30 LLINs will be withdrawn for bio-efficacy and chemical analysis every 6 months up to 36 months and another 30 collected for experimental hut trials every year. Bio-efficacy will be assessed using cone bioassays and tunnel tests against susceptible and resistant laboratory strains of Anopheles gambiae sensu stricto. Efficacy of field-collected nets will be compared in six experimental huts. The main outcomes will be Anopheles mortality up to 72 h post exposure, blood feeding and egg maturation using ovary dissection to assess impact on fecundity. Conclusions Study findings will help develop bio-efficacy and physical durability criteria for partner A.I., in relation to the cRCT epidemiological and entomological outcomes, and refine preferred product characteristics of each class of LLIN. If suitable, the bioassay and hut outcomes will be fitted to transmission models to estimate correlation with cRCT outcomes. Trial registration number: NCT03554616.

Abstract Background Indoor residual spraying (IRS) is a major method of malaria vector control across sub-Saharan Africa. Effective control is being undermined by the rapid spread of insecticide resistance. There is major investment in development of new insecticides for IRS that possess novel modes of action, long residual activity, low mammalian toxicity and minimal cross-resistance. VECTRON™ T500, a new IRS product containing the active ingredient broflanilide as a 50% wettable powder (WP), has been shown to be efficacious against pyrethroid susceptible and resistant vector species on mud and concrete substrates in experimental hut (Phase II) trials. Methods A two-arm non-inferiority cluster randomized controlled trial (Phase III) will be undertaken in Muheza District, Tanga Region, Tanzania. VECTRON™ T500 will be compared to the IRS product Fludora® Fusion (clothianidin 50% WP + deltamethrin 6.25% WP). The predominant malaria vectors in the study area are pyrethroid-resistant Anopheles gambiae s.s., An. arabiensis and An. funestus s.s. Sixteen village clusters will be pair-matched on baseline vector densities and allocated to reference and intervention arms. Consenting households in the intervention arm will be sprayed with VECTRON™ T500 and those in the reference arm will be sprayed with Fludora® Fusion. Each month, CDC light traps will collect mosquitoes to estimate changes in vector density, indoor biting, sporozoite and entomological inoculation rates (EIR). Susceptibility to IRS active ingredients will be assessed using World Health Organisation (WHO) bottle bioassays. Target site and metabolic resistance mechanisms will be characterised among Anopheles field populations from both trial arms. Residual efficacy of both IRS products will be monitored for 12 months post intervention. Questionnaire and focus group discussions will explore factors that influence adherence, adverse effects and benefits of IRS. Discussion This protocol describes a large-scale non-inferiority evaluation of a novel IRS product to reduce the density and EIR of pyrethroid-resistant Anopheles vectors. If VECTRON™ T500 proves non-inferior to Fludora® Fusion, it will be considered as an additional vector control product for malaria prevention and insecticide resistance management. Trial registration: ClinicalTrials.gov, NCT05150808, registered on 26 November 2021. Retrospectively registered.

Abstract Background Malaria vector control in the Democratic Republic of the Congo is plagued by several major challenges, including inadequate infrastructure, lack of access to health care systems and preventative measures, and more recently the widespread emergence of insecticide resistance among Anopheles mosquitoes. Across 26 provinces, insecticide resistance has been reported from multiple sentinel sites. However, to date, investigation of molecular resistance mechanisms among Anopheles vector populations in DRC has been more limited. Methods Adult Anopheles gambiae sensu lato (s.l.) and Anopheles funestus s.l. were collected from two sites in Sud-Kivu province and one site in Haut-Uélé province and PCR-screened for the presence of 11 resistance mutations, to provide additional information on frequency of resistance mechanisms in the eastern DRC, and to critically evaluate the utility of these markers for prospective country-wide resistance monitoring. Results L1014F-kdr and L1014S-kdr were present in 75.9% and 56.7% of An. gambiae s.l. screened, respectively, with some individuals harbouring both resistant alleles. Across the three study sites, L43F-CYP4J5 allele frequency ranged from 0.42 to 0.52, with evidence for ongoing selection. G119S-ace1 was also identified in all sites but at lower levels. A triple mutant haplotype (comprising the point mutation CYP6P4-I236M, the insertion of a partial Zanzibar-like transposable element and duplication of CYP6AA1) was present at high frequencies. In An. funestus s.l. cis-regulatory polymorphisms in CYP6P9a and CYP6P9b were detected, with allele frequencies ranging from 0.82 to 0.98 and 0.65 to 0.83, respectively. Conclusions This study screened the most up-to-date panel of DNA-based resistance markers in An. gambiae s.l. and An. funestus s.l. from the eastern DRC, where resistance data is lacking. Several new candidate markers (CYP4J5, G119S-ace1, the triple mutant, CYP6P9a and CYP6P9b) were identified, which are diagnostic of resistance to major insecticide classes, and warrant future, larger-scale monitoring in the DRC to inform vector control decisions by the National Malaria Control Programme.

Abstract Background Despite widespread use of long-lasting insecticidal nets (LLINs) and other tools, malaria caused 409,000 deaths worldwide in 2019. While indoor residual spraying (IRS) is an effective supplement, IRS is moderately expensive and logistically challenging. In endemic areas, IRS requires yearly application just before the main rainy season and potential interim reapplications. A new technology, insecticide-treated wall liner (ITWL), might overcome these challenges. Methods We conducted a 44-cluster two-arm randomized controlled trial in Muheza, Tanzania from 2015 to 2016 to evaluate the cost and efficacy of a non-pyrethroid ITWL to supplement LLINs, analyzing operational changes over three installation phases. The estimated efficacy (with 95% confidence intervals) of IRS as a supplement to LLINs came mainly from a published randomized trial in Muleba, Tanzania. We obtained financial costs of IRS from published reports and conducted a household survey of a similar IRS program near Muleba to determine household costs. The costs of ITWL were amortized over its 4-year expected lifetime and converted to 2019 US dollars using Tanzania’s GDP deflator and market exchange rates. Results Operational improvements from phases 1 to 3 raised ITWL coverage from 35.1 to 67.1% of initially targeted households while reducing economic cost from $34.18 to $30.56 per person covered. However, 90 days after installing ITWL in 5666 households, the randomized trial was terminated prematurely because cone bioassay tests showed that ITWL no longer killed mosquitoes and therefore could not prevent malaria. The ITWL cost $10.11 per person per year compared to $5.69 for IRS. With an efficacy of 57% (3–81%), IRS averted 1162 (61–1651) disability-adjusted life years (DALYs) per 100,000 population yearly. Its incremental cost-effectiveness ratio (ICER) per DALY averted was $490 (45% of Tanzania’s per capita gross national income). Conclusions These findings provide design specifications for future ITWL development and implementation. It would need to be efficacious and more effective and/or less costly than IRS, so more persons could be protected with a given budget. The durability of a previous ITWL, progress in non-pyrethroid tools, economies of scale and competition (as occurred with LLINs), strengthened community engagement, and more efficient installation and management procedures all offer promise of achieving these goals. Therefore, ITWLs merit ongoing study. First posted 2015 ( NCT02533336 ).

Pyriproxyfen (PPF) is an insect growth regulator used in the co-treatment of long-lasting insecticidal nets for its ability to sterilize female mosquitoes. To evaluate the efficacy of PPF-treated nets on mosquito reproductivity, most studies observe oviposition (egg-laying) rates in the laboratory. This technique has several technical disadvantages. Our study assessed if ovarial dissection could serve as an effective proxy for evaluating sterility in Anopheles gambiae mosquitoes. Blood-fed females were exposed to untreated or PPF-treated nets in cylinder assays and followed over several days to observe oviposition rates or egg development by dissection. For identifying PPF-exposed mosquitoes, both techniques demonstrated high sensitivity (oviposition: 99.1%; dissection: 100.0%), but for identifying non-exposed mosquitoes, specificity was significantly higher in the dissection group (52.5% vs. 18.9%). To assess whether dissection could be applied to nets treated with a pyrethroid or co-treated with a pyrethroid and PPF in tunnel tests, a blinded investigator performed dissections to predict the PPF exposure status across different treatment groups. The exposure status of dissected females was predicted with >90% accuracy. We report that dissection is a sensitive technique to assess sterility in female Anopheles gambiae mosquitoes and can be used as a predictor of PPF exposure.

The efficacy of a vector control tool in reducing mosquito biting is crucial for its acceptability. The present study compared the vector density of Culex spp. And Mansonia spp. across clusters, which received two dual-active ingredient (a.i.) long-lasting insecticidal nets (LLINs) and a standard pyrethroid-only LLIN, and assessed the seasonality of these mosquito genera. A total of 85,723 Culex spp. and 144,025 Mansonia spp. were caught over the study period. The density of Culex and Mansonia was reduced in all three arms over the study period. There was no evidence of a significant reduction in the indoor or outdoor density of Culex spp. in either dual-a.i. LLIN arm as compared to the standard pyrethroid-only net arm. A similar trend was observed with Mansonia spp. A high density of Culex spp. was found both in rainy and dry seasons, while for Mansonia spp., this was mainly observed during the rainy season. These results suggest that the novel insecticides in the dual-a.i. LLINs did not have an additional impact on these species and that pyrethroids might still be effective on them. Further work is required to determine whether these species of mosquitoes have resistance to the insecticides tested in this trial.

Abstract Anopheles funestus is playing an increasing role in malaria transmission in parts of sub-Saharan Africa, where An. gambiae s.s. has been effectively controlled by long-lasting insecticidal nets. We investigated vector population bionomics, insecticide resistance and malaria transmission dynamics in 86 study clusters in North-West Tanzania. An. funestus s.l. represented 94.5% (4740/5016) of all vectors and was responsible for the majority of malaria transmission (96.5%), with a sporozoite rate of 3.4% and average monthly entomological inoculation rate (EIR) of 4.57 per house. Micro-geographical heterogeneity in species composition, abundance and transmission was observed across the study district in relation to key ecological differences between northern and southern clusters, with significantly higher densities, proportions and EIR of An. funestus s.l. collected from the South. An. gambiae s.l. (5.5%) density, principally An. arabiensis (81.1%) and An. gambiae s.s. (18.9%), was much lower and closely correlated with seasonal rainfall. Both An. funestus s.l. and An. gambiae s.l. were similarly resistant to alpha-cypermethrin and permethrin. Overexpression of CYP9K1, CYP6P3, CYP6P4 and CYP6M2 and high L1014S-kdr mutation frequency were detected in An. gambiae s.s. populations. Study findings highlight the urgent need for novel vector control tools to tackle persistent malaria transmission in the Lake Region of Tanzania.

Anopheles stephensi mosquitoes, efficient vectors in parts of Asia and Africa, were found in 75.3% of water sources surveyed and contributed to 80.9% of wild-caught Anopheles mosquitoes in Awash Sebat Kilo, Ethiopia. High susceptibility of these mosquitoes to Plasmodium falciparum and vivax infection presents a challenge for malaria control in the Horn of Africa.

Abstract Background Long-lasting insecticidal nets (LLINs) are currently the primary method of malaria control in sub-Saharan Africa and have contributed to a significant reduction in malaria burden over the past 15 years. However, this progress is threatened by the wide-scale selection of insecticide-resistant malaria vectors. It is, therefore, important to accelerate the generation of evidence for new classes of LLINs. Methods This protocol presents a three-arm superiority, single-blinded, cluster randomized controlled trial to evaluate the impact of 2 novel dual-active ingredient LLINs on epidemiological and entomological outcomes in Benin, a malaria-endemic area with highly pyrethroid-resistant vector populations. The study arms consist of (i) Royal Guard® LLIN, a net combining a pyrethroid (alpha-cypermethrin) plus an insect growth regulator (pyriproxyfen), which in the adult female is known to disrupt reproduction and egg fertility; (ii) Interceptor G2® LLIN, a net incorporating two adulticides (alpha-cypermethrin and chlorfenapyr) with different modes of action; and (iii) the control arm, Interceptor® LLIN, a pyrethroid (alpha-cypermethrin) only LLIN. In all arms, one net for every 2 people will be distributed to each household. Sixty clusters were identified and randomised 1:1:1 to each study arm. The primary outcome is malaria case incidence measured over 24 months through active case detection in a cohort of 25 children aged 6 months to 10 years, randomly selected from each cluster. Secondary outcomes include 1) malaria infection prevalence (all ages) and prevalence of moderate to severe anaemia in children under 5 years old, measured at 6 and 18 months post-intervention; 2) entomological indices measured every 3 months using human landing catches over 24 months. Insecticide resistance intensity will also be monitored over the study period. Discussion This study is the second cluster randomised controlled trial to evaluate the efficacy of these next-generation LLINs to control malaria transmitted by insecticide-resistant mosquitoes. The results of this study will form part of the WHO evidence-based review to support potential public health recommendations of these nets and shape malaria control strategies of sub-Saharan Africa for the next decade. Trial registration ClinicalTrials.gov, NCT03931473 , registered on 30 April 2019.

Entomological surveillance in Benin has historically been limited to zones where indoor residual spraying was performed or where long-standing sentinel surveillance sites existed. However, there are significant country-wide gaps in entomological knowledge. The National Malaria Control Program (NMCP) assessed population dynamics of Anopheles vectors and malaria transmission in each of Benin’s 12 departments to create an entomological risk profile. Two communes per department (24/77 communes) were chosen to reflect diverse geographies, ecologies and malaria prevalence. Two villages per commune were selected from which four households (HH) per village were used for human landing catches (HLCs). In each HH, an indoor and outdoor HLC occurred between 7 p.m. and 7 a.m. on two consecutive nights between July–September 2017. Captured Anopheles were identified, and ovaries were dissected to determine parous rate. Heads and thoraces were tested for Plasmodium falciparum sporozoites by ELISA. The Entomological Inoculation Rate (EIR) was calculated as the product of mosquito bite rate and sporozoite index. Bite rates from An. gambiae s.l., the primary vector species complex, differed considerably between communes; average sporozoite infection index was 3.5%. The EIR ranged from 0.02 infectious bites (ib) per human per night in the departments of Ouémé and Plateau to 1.66 ib/human/night in Collines. Based on transmission risk scales, Avrankou, Sakété and Nikki are areas of low transmission (0 < EIR < 3 ib/human/year), Adjarra, Adja Ouèrè, Zè, Toffo, Bopa, Pehunco, Pèrèrè and Kandi are of medium transmission (3 < EIR < 30 ib/human/year), and the other remaining districts are high transmission (EIR > 30 ib/human/year). The heterogeneous and diverse nature of malaria transmission in Benin was not readily apparent when only assessing entomological surveillance from sentinel sites. Prospectively, the NMCP will use study results to stratify and deploy targeted vector control interventions in districts with high EIRs to better protect populations most at-risk.

Metacyclogenesis is one of the most important processes in the life cycle of Trypanosoma cruzi. In this stage, noninfective epimastigotes become infective metacyclic trypomastigotes. However, the transcriptomic changes that occur during this transformation remain uncertain. Illumina RNA-sequencing of epimastigotes and metacyclic trypomastigotes belonging to T. cruzi DTU I was undertaken. Sequencing reads were aligned and mapped against the reference genome, differentially expressed genes between the two life cycle stages were identified, and metabolic pathways were reconstructed. Gene expression differed significantly between epimastigotes and metacyclic trypomastigotes. The cellular pathways that were mostly downregulated during metacyclogenesis involved glucose energy metabolism (glycolysis, pyruvate metabolism, the Krebs cycle, and oxidative phosphorylation), amino acid metabolism, and DNA replication. By contrast, the processes where an increase in gene expression was observed included those related to autophagy (particularly Atg7 and Atg8 transcripts), corroborating its importance during metacyclogenesis, endocytosis, by an increase in the expression of the AP-2 complex subunit alpha, protein processing in the endoplasmic reticulum and meiosis. Study findings indicate that in T. cruzi metacyclic trypomastigotes, metabolic processes are decreased, and expression of genes involved in specific cell cycle processes is increased to facilitate transformation to this infective stage.

Two billion pyrethroid long-lasting insecticidal nets (LLINs) have been distributed since 2004 for malaria prevention in Sub-Saharan Africa. Current malaria control strategies rely on an assumed effective 3-year lifespan for LLINs. PBO synergist LLINs are a newly recommended class of net but there is limited information on their life span and long-term protective efficacy in communities. To assess their operational survival, a cohort of 390 PBO LLINs (Olyset Plus) and 367 standard pyrethroid LLIN (Olyset net) from 396 households were followed for 36 months in Western Tanzania. To assess the association between the condition of the LLIN and malaria infection, nets from at least 480 randomly selected households were assessed during malaria prevalence cross-sectional surveys at 4, 9, 16, 21, 28, and 33 months post-distribution. Information on the presence and condition of nets, and demographic information from the household, were collected to evaluate factors influencing net durability. After 3 years less than 17% of nets distributed still remained in the households. The fabric condition was not associated with malaria infection in either type of net. The difference between the net types was highest when nets were between 1–2 years old, when PBO nets appeared to be similarly protective as nets less than a year old, whereas standard nets were considerably less protective as they aged, regardless of fabric condition. There was no statistical difference in the estimated median functional survival time between net types with 1.6 years (95% CI 1.38–1.87) for PBO LLIN and 1.9 years (95% CI 1.67–2.06) for standard LLINs. After 3 years, there was a loss of 55% of permethrin (pyrethroid) content for both nets, and 97% of PBO content was lost in PBO LLIN. These results highlight that functional survival is less than the recommended 3 years for both net types. However, even as the nets age, the PBO nets remained more protective than standard nets, regardless of their condition.

Abstract Background Between 2011 and 2018, an estimated 134.8 million pyrethroid-treated long-lasting insecticidal nets (LLINs) were distributed nationwide in the Democratic Republic of Congo (DRC) for malaria control. Pyrethroid resistance has developed in DRC in recent years, but the intensity of resistance and impact on LLIN efficacy was not known. Therefore, the intensity of resistance of Anopheles gambiae sensu lato (s.l.) to permethrin and deltamethrin was monitored before and after a mass distribution of LLINs in Kinshasa in December 2016, and in 6 other sites across the country in 2017 and 11 sites in 2018. Methods In Kinshasa, CDC bottle bioassays using 1, 2, 5, and 10 times the diagnostic dose of permethrin and deltamethrin were conducted using An. gambiae s.l. collected as larvae and reared to adults. Bioassays were conducted in four sites in Kinshasa province 6 months before a mass distribution of deltamethrin-treated LLINs and then two, six, and 10 months after the distribution. One site in neighbouring Kongo Central province was used as a control (no mass campaign of LLIN distribution during the study). Nationwide intensity assays were conducted in six sites in 2017 using CDC bottle bioassays and in 11 sites in 2018 using WHO intensity assays. A sub-sample of An. gambiae s.l. was tested by PCR to determine species composition and frequency of kdr-1014F and 1014S alleles. Results In June 2016, before LLIN distribution, permethrin resistance intensity was high in Kinshasa; the mean mortality rate was 43% at the 5× concentration and 73% at the 10× concentration. Bioassays at 3 time points after LLIN distribution showed considerable variation by site and time and there was no consistent evidence for an increase in pyrethroid resistance intensity compared to the neighbouring control site. Tests of An. gambiae s.l. in 6 sites across the country in 2017 and 11 sites in 2018 showed all populations were resistant to the diagnostic doses of 3 pyrethroids. In 2018, the intensity of resistance varied by site, but was generally moderate for all three pyrethroids, with survivors at ×5 the diagnostic dose. Anopheles gambiae sensu stricto (s.s.) was the most common species identified across 11 sites in DRC, but in Kinshasa, An. gambiae s.s. (91%) and Anopheles coluzzii (8%) were sympatric. Conclusions Moderate or high intensity pyrethroid resistance was detected nationwide in DRC and is a serious threat to sustained malaria control with pyrethroid LLINs. Next generation nets (PBO nets or bi-treated nets) should be considered for mass distribution.

Abstract Background Several mosquito collection methods are routinely used in vector control programmes. However, they target different behaviours causing bias in estimation of species diversity and abundance. Given the paucity of mosquito trap data in West Africa, we compared the performance of five trap-lure combinations and Human Landing Catches (HLCs) in Guinea. Methods CDC light traps (LT), BG sentinel 2 traps (BG2T), gravid traps (GT) and Stealth traps (ST) were compared in a 5 × 5 Latin Square design in three villages in Guinea between June and July 2018. The ST, a portable trap which performs similarly to a LT but incorporates LEDs and incandescent light, was included since it has not been widely tested. BG2T were used with BG and MB5 lures instead of CO2 to test the efficacy of these attractants. HLCs were performed for 5 nights, but not as part of the Latin Square. A Generalised Linear Mixed Model was applied to compare the effect of the traps, sites and collection times on mosquito abundance. Species identification was confirmed using PCR-based analysis and Sanger sequencing. Results A total of 10,610 mosquitoes were captured across five traps. ST collected significantly more mosquitoes (7096) than the rest of the traps, but resulted in a higher number of damaged specimens. ST and BG2T collected the highest numbers of Anopheles gambiae (s.l.) and Aedes aegypti mosquitoes, respectively. HLCs captured predominantly An. coluzzii (41%) and hybrids of An. gambiae and An. coluzzii (36%) in contrast to the five traps, which captured predominantly An. melas (83%). The rural site (Senguelen) presented the highest abundance of mosquitoes and overall diversity in comparison with Fandie (semi-rural) and Maferinyah Centre I (semi-urban). Our results confirm the presence of four species for the first time in Guinea. Conclusions ST collected the highest number of mosquitoes suggesting this trap may play an important role for mosquito surveillance in Guinea and similar sites in West Africa. We recommend the incorporation of molecular tools in entomological studies since they have helped to identify 25 mosquito species in this area.

Abstract Background Insecticide resistance is a growing threat to malaria vector control. Ivermectin, either administered to humans or animals, may represent an alternate strategy to reduce resistant mosquito populations. The aim of this study was to assess the residual or delayed effect of administering a single oral dose of ivermectin to humans on the survival, fecundity and fertility of Anopheles arabiensis in Ethiopia. Methods Six male volunteers aged 25–40 years (weight range 64–72 kg) were recruited; four of them received a recommended single oral dose of 12 mg ivermectin and the other two individuals were untreated controls. A fully susceptible insectary colony of An. arabiensis was fed on treated and control participants at 1, 4, 7, 10 and 13 days post ivermectin-administration. Daily mosquito mortality was recorded for 5 days. An. arabiensis fecundity and fertility were measured from day 7 post treatment, by dissection to examine the number of eggs per mosquito, and by observing larval hatching rates, respectively. Results Ivermectin treatment induced significantly higher An. arabiensis mortality on days 1 and 4, compared to untreated controls (p = 0.02 and p

ABSTRACT Insecticide resistance among mosquito species is now a pervasive phenomenon that threatens to jeopardize global malaria vector control efforts. Evidence of links between the mosquito microbiota and insecticide resistance is emerging, with significant enrichment of insecticide degrading bacteria and enzymes in resistant populations. Using 16S rRNA amplicon sequencing, we characterized and compared the microbiota of Anopheles coluzzii in relation to their deltamethrin resistance and exposure profiles. Comparisons between 2- and 3-day-old deltamethrin-resistant and -susceptible mosquitoes demonstrated significant differences in microbiota diversity. Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera, each of which comprised insecticide-degrading species, were significantly enriched in resistant mosquitoes. Susceptible mosquitoes had a significant reduction in alpha diversity compared to resistant individuals, with Asaia and Serratia dominating microbial profiles. There was no significant difference in deltamethrin-exposed and -unexposed 5- to 6-day-old individuals, suggesting that insecticide exposure had minimal impact on microbial composition. Serratia and Asaia were also dominant in 5- to 6-day-old mosquitoes, which had reduced microbial diversity compared to 2- to 3-day-old mosquitoes. Our findings revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for insecticide resistance surveillance. qPCR detection of Serratia and Asaia was consistent with 16S rRNA sequencing, suggesting that population-level field screening of bacterial microbiota may be feasibly integrated into wider resistance monitoring, if reliable and reproducible markers associated with phenotype can be identified. IMPORTANCE Control of insecticide-resistant vector populations remains a significant challenge to global malaria control and while substantial progress has been made elucidating key target site mutations, overexpressed detoxification enzymes and alternate gene families, the contribution of the mosquito microbiota to phenotypic insecticide resistance has been largely overlooked. We focused on determining the effects of deltamethrin resistance intensity on Anopheles coluzzii microbiota and identifying any microbial taxa associated with phenotype. We demonstrated a significant reduction in microbial diversity between deltamethrin-resistant and -susceptible mosquitoes. Insecticide degrading bacterial species belonging to Ochrobactrum, Lysinibacillus, and Stenotrophomonas genera were significantly enriched in resistant mosquitoes, while Asaia and Serratia dominated microbial profiles of susceptible individuals. Our results revealed significant alterations of Anopheles coluzzii microbiota associated with deltamethrin resistance, highlighting the potential for identification of novel microbial markers for surveillance and opportunities for designing innovative control techniques to prevent the further evolution and spread of insecticide resistance.

Dengue Fever (DF) is an important arthropod-borne viral infection that has repeatedly occurred as outbreaks in eastern and northeastern Ethiopia since 2013. A cross-sectional epidemiological outbreak investigation was carried out from September to November 2019 on febrile patients (confirmed malaria negative) who presented with suspected and confirmed DF at both public and private health facilities in Gewane District, Afar Region, northeastern Ethiopia. Entomological investigation of containers found in randomly selected houses belonging to DF-positive patients was undertaken to survey for the presence of Aedes larvae/pupae. A total of 1185 DF cases were recorded from six health facilities during the 3-month study period. The mean age of DF cases was 27.2 years, and 42.7% of cases were female. The most affected age group was 15–49 years old (78.98%). The total case proportions differed significantly across age groups when compared to the population distribution; there were approximately 15% and 5% higher case proportions among those aged 15–49 years and 49+ years, respectively. A total of 162 artificial containers were inspected from 62 houses, with 49.4% found positive for Aedes aegypti larva/pupae. Aedes mosquitoes were most commonly observed breeding in plastic tanks, tires, and plastic or metal buckets/bowls. World Health Organization entomological indices classified the study site as high risk for dengue virus outbreaks (House Index = 45.2%, Container Index = 49.4%, and Breteau Index = 129). Time series climate data, specifically rainfall, were found to be significantly predictive of AR (p = 0.035). Study findings highlight the importance of vector control to prevent future DF outbreaks in the region. The scarcity of drinking water and microclimatic conditions may have also contributed to the occurrence of this outbreak.

Abstract Background In recent years, the scale-up of long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) has greatly reduced malaria transmission. However, malaria remains a global public health concern with the majority of the disease burden in sub-Saharan Africa. Insecticide resistance is a growing problem among Anopheles vector populations, with potential implications for the continued effectiveness of available control interventions. Improved understanding of current resistance levels and underlying mechanisms is essential to design appropriate management strategies and to mitigate future selection for resistance. Methods Anopheles gambiae sensu lato mosquitoes were collected from three villages in Faranah Prefecture, Guinea and their levels of susceptibility to seven insecticides were measured using CDC resistance intensity bioassays. Synergist assays with piperonyl butoxide (PBO) were also undertaken to assess the role of elevated mixed-function oxidases in resistance. Five hundred and sixty-three mosquitoes underwent molecular characterization of vector species, presence of target site mutations (L1014F kdr, N1575Y and G119S Ace-1), Plasmodium falciparum infection, and relative expression of three metabolic genes (CYP6M2, CYP6P3 and GSTD3). Results In Faranah, resistance to permethrin and deltamethrin was observed, as well as possible resistance to bendiocarb. All assayed vector populations were fully susceptible to alpha-cypermethrin, pirimiphos-methyl, clothianidin and chlorfenapyr. Plasmodium falciparum infection was detected in 7.3% (37/508) of mosquitoes tested. The L1014F kdr mutation was found in 100% of a sub-sample of 60 mosquitoes, supporting its fixation in the region. The N1575Y mutation was identified in 20% (113/561) of individuals, with ongoing selection evidenced by significant deviations from Hardy–Weinberg equilibrium. The G119S Ace-1 mutation was detected in 62.1% (18/29) of mosquitoes tested and was highly predictive of bendiocarb bioassay survival. The metabolic resistance genes, CYP6M2, CYP6P3 and GSTD3, were found to be overexpressed in wild resistant and susceptible An. gambiae sensu stricto populations, compared to a susceptible G3 colony. Furthermore, CYP6P3 was significantly overexpressed in bendiocarb survivors, implicating its potential role in carbamate resistance in Faranah. Conclusions Identification of intense resistance to permethrin and deltamethrin in Faranah, is of concern, as the Guinea National Malaria Control Programme (NMCP) relies exclusively on the distribution of pyrethroid-treated LLINs for vector control. Study findings will be used to guide current and future control strategies in the region.

Abstract Insecticide resistance across sub-Saharan Africa may impact the continued effectiveness of malaria vector control. We investigated the association between carbamate and pyrethroid resistance with Anopheles gambiae s.l. parity, Plasmodium falciparum infection, and molecular insecticide resistance mechanisms in Guinea. Pyrethroid resistance was intense, with field populations surviving ten times the insecticidal concentration required to kill susceptible individuals. The L1014F kdr-N1575Y haplotype and I1527T mutation were significantly associated with mosquito survival following permethrin exposure (Prevalence Ratio; PR = 1.92, CI = 1.09–3.37 and PR = 2.80, CI = 1.03–7.64, respectively). Partial restoration of pyrethroid susceptibility following synergist pre-exposure suggests a role for mixed-function oxidases. Carbamate resistance was lower and significantly associated with the G119S Ace-1 mutation. Oocyst rates were 6.8% and 4.2% among resistant and susceptible mosquitoes, respectively; survivors of bendiocarb exposure were significantly more likely to be infected. Pyrethroid resistant mosquitoes had significantly lower parity rates than their susceptible counterparts (PR = 1.15, CI = 1.10–1.21). Our findings emphasize the need for additional studies directly assessing the influence of insecticide resistance on mosquito fitness.

Abstract Background Insecticide-based interventions play an integral role in malaria vector control. However, the continued spread of insecticide resistance threatens to undermine progress made thus far and may ultimately lead to operational failure of current control measures. Clothianidin and chlorfenapyr both have unique modes of action and have expanded the number of insecticide classes available to vector control programmes. Prior to field use, it is imperative to establish their toxicity against local mosquito populations and evaluate potential cross-resistance with other chemicals used contemporarily or historically. The aim of this study was to determine the diagnostic doses of clothianidin and chlorfenapyr and their efficacies against Anopheles arabiensis, the predominant Ethiopian malaria vector species. Methods A range of doses of clothianidin and chlorfenapyr were tested, using modified WHO susceptibility tests and CDC bottle bioassays, respectively, against an Ethiopian susceptible laboratory strain and a wild population of An. arabiensis collected from Oromia Region, Ethiopia. Cross-resistance to other public health insecticides: carbamates (bendiocarb and propoxur), organophosphate (malathion) and pyrethroids (deltamethrin and permethrin), was assessed in the same mosquito populations using CDC bottle bioassays. Results Complete mosquito mortality was observed with the laboratory strain using the recommended diagnostic doses for clothianidin (2%/filter paper) and chlorfenapyr (100 µg/bottle). The field population was resistant to malathion (83% mortality), capable of surviving 2×, 5× and 10× the diagnostic dose of both deltamethrin and permethrin, but susceptible to bendiocarb and propoxur. The field population of An. arabiensis was significantly more susceptible to clothianidin, reaching 100% mortality by day 2 compared to the laboratory strain (100% mortality by day 3). In contrast, the wild population was less susceptible to chlorfenapyr, with the highest mortality of 99% at 72 h using 200 µg/bottle compared to the laboratory colony, which reached complete mortality at 50 µg/bottle by 24 h. Conclusions The putative diagnostic doses of clothianidin and chlorfenapyr are appropriate for monitoring resistance in An. arabiensis from Ethiopia. The unique modes of action and an absence of cross-resistance render clothianidin and chlorfenapyr potential candidates for inclusion in the National Malaria Control Programme vector control efforts, particularly in areas with high pre-existing or emergent resistance to other insecticide classes.

Durability monitoring of insecticide-treated nets (ITNs) containing a pyrethroid in combination with a second active ingredient (AI) must be adapted so that the insecticidal bioefficacy of each AI can be monitored independently. An effective way to do this is to measure rapid knock down of a pyrethroid-susceptible strain of mosquitoes to assess the bioefficacy of the pyrethroid component and to use a pyrethroid-resistant strain to measure the bioefficacy of the second ingredient. To allow robust comparison of results across tests within and between test facilities, and over time, protocols for bioefficacy testing must include either characterisation of the resistant strain, standardisation of the mosquitoes used for bioassays, or a combination of the two. Through a series of virtual meetings, key stakeholders and practitioners explored different approaches to achieving these goals. Via an iterative process we decided on the preferred approach and produced a protocol consisting of characterising mosquitoes used for bioefficacy testing before and after a round of bioassays, for example at each time point in a durability monitoring study. We present the final protocol and justify our approach to establishing a standard methodology for durability monitoring of ITNs containing pyrethroid and a second AI.

Trypanosoma cruzi, a zoonotic kinetoplastid protozoan parasite, is the causative agent of American trypanosomiasis (Chagas disease). Having a very plastic, repetitive and complex genome, the parasite displays a highly diverse repertoire of surface molecules, with pivotal roles in cell invasion, immune evasion and pathogenesis. Before 2016, the complexity of the genomic regions containing these genes impaired the assembly of a genome at chromosomal level, making it impossible to study the structure and function of the several thousand repetitive genes encoding the surface molecules of the parasite. We here describe the genome assembly of the Sylvio X10/1 genome sequence, which since 2016 has been used as a reference genome sequence for T. cruzi clade I (TcI), produced using high coverage PacBio single-molecule sequencing. It was used to analyze deep Illumina sequence data from 34 T. cruzi TcI isolates and clones from different geographic locations, sample sources and clinical outcomes. Resolution of the surface molecule gene distribution showed the unusual duality in the organization of the parasite genome, a synteny of the core genomic region with related protozoa flanked by unique and highly plastic multigene family clusters encoding surface antigens. The presence of abundant interspersed retrotransposons in these multigene family clusters suggests that these elements are involved in a recombination mechanism for the generation of antigenic variation and evasion of the host immune response on these TcI strains. The comparative genomic analysis of the cohort of TcI strains revealed multiple cases of such recombination events involving surface molecule genes and has provided new insights into T. cruzi population structure.

Introduction The massive scale-up of long-lasting insecticidal nets (LLINs) has led to major reductions in malaria burden in many sub-Saharan African countries. This progress is threatened by widespread insecticide resistance among malaria vectors. This cluster-randomised controlled trial (c-RCT) compares three of the most promising dual active ingredients LLINs (dual-AI LLINs), which incorporate mixtures of insecticides or insecticide synergists to standard LLINs in an area of pyrethroid insecticide resistance.Methods A four-arm, single-blinded, c-RCT will evaluate the effectiveness of three types of dual-AI LLINs (1) Royal Guard, combining two insecticides, pyriproxyfen and the pyrethroid alpha-cypermethrin; (2) Interceptor G2, combining chlorfenapyr and alpha-cypermethrin; (3) Olyset Plus, an LLIN combining a synergist, piperonyl butoxide and the pyrethroid permethrin, compared with; (4) Interceptor LN, a standard LLIN containing the pyrethroid alpha-cypermethrin as the sole AI. The primary outcomes are malaria infection prevalence in children aged 6 months–14 years and entomological inoculation rate (EIR), as a standard measure of malaria transmission at 24 months postintervention and cost-effectiveness.Ethics and dissemination Ethical approval was received from the institutional review boards of the Tanzanian National Institute for Medical Research, Kilimanjaro Christian Medical University College, London School of Hygiene and Tropical Medicine, and University of Ottawa. Study findings will be actively disseminated via reports and presentations to stakeholders, local community leaders, and relevant national and international policy makers as well as through conferences, and peer-reviewed publications.Trial registration number NCT03554616.

In response to growing concerns over the sustained effectiveness of pyrethroid-only based control tools, new products are being developed and evaluated. Some examples of these are dual-active ingredient (AI) insecticide-treated nets (ITNs) which contain secondary insecticides, or synergist ITNs which contain insecticide synergist, both in combination with a pyrethroid. These net types are often termed ‘next-generation’ insecticide-treated nets. Several of these new types of ITNs are being evaluated in large-scale randomized control trials (RCTs) and pilot deployment schemes at a country level. However, no methods for measuring the biological durability of the AIs or synergists on these products are currently recommended. In this publication, we describe a pipeline used to collate and interrogate several different methods to produce a singular ‘consensus standard operating procedure (SOP)’, for monitoring the biological durability of three new types of ITNs: pyrethroid + piperonyl butoxide (PBO), pyrethroid + pyriproxyfen (PPF), and pyrethroid + chlorfenapyr (CFP). This process, convened under the auspices of the Innovation to Impact programme, sought to align methodologies used for conducting durability monitoring activities of next-generation ITNs.

Abstract Background Indoor residual spraying (IRS) and long-lasting insecticidal nets (LLINs) remain the cornerstones of malaria vector control. However, the development of insecticide resistance and its implications for operational failure of preventative strategies are of concern. The aim of this study was to characterize insecticide resistance among Anopheles arabiensis populations in Ethiopia and describe temporal and spatial patterns of resistance between 2012 and 2016. Methods Between 2012 and 2016, resistance status of An. arabiensis was assessed annually during the long rainy seasons in study sites from seven of the nine regions in Ethiopia. Insecticide resistance levels were measured with WHO susceptibility tests and CDC bottle bioassays using insecticides from four chemical classes (organochlorines, pyrethroids, organophosphates and carbamates), with minor variations in insecticides tested and assays conducted between years. In selected sites, CDC synergist assays were performed by pre-exposing mosquitoes to piperonyl butoxide (PBO). In 2015 and 2016, mosquitoes from DDT and deltamethrin bioassays were randomly selected, identified to species-level and screened for knockdown resistance (kdr) by PCR. Results Intense resistance to DDT and pyrethroids was pervasive across Ethiopia, consistent with historic use of DDT for IRS and concomitant increases in insecticide-treated net coverage over the last 15 years. Longitudinal resistance trends to malathion, bendiocarb, propoxur and pirimiphos-methyl corresponded to shifts in the national insecticide policy. By 2016, resistance to the latter two insecticides had emerged, with the potential to jeopardize future long-term effectiveness of vector control activities in these areas. Between 2015 and 2016, the West African (L1014F) kdr allele was detected in 74.1% (n = 686/926) of specimens, with frequencies ranging from 31 to 100% and 33 to 100% in survivors from DDT and deltamethrin bioassays, respectively. Restoration of mosquito susceptibility, following pre-exposure to PBO, along with a lack of association between kdr allele frequency and An. arabiensis mortality rate, both indicate metabolic and target-site mutation mechanisms are contributing to insecticide resistance. Conclusions Data generated by this study will strengthen the National Malaria Control Programme’s insecticide resistance management strategy to safeguard continued efficacy of IRS and other malaria control methods in Ethiopia.

Abstract While long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS) are the cornerstones of malaria vector control throughout sub-Saharan Africa, there is an urgent need for the development of novel insecticide delivery mechanisms to sustain and consolidate gains in disease reduction and to transition towards malaria elimination and eradication. Insecticide-treated durable wall lining (ITWL) may represent a new paradigm for malaria control as a potential complementary or alternate longer-lasting intervention to IRS. ITWL can be attached to inner house walls, remain efficacious over multiple years and overcome some of the operational constraints of first-line control strategies, specifically nightly behavioural compliance required of LLINs and re-current costs and user fatigue associated with IRS campaigns. Initial experimental hut trials of insecticide-treated plastic sheeting reported promising results, achieving high levels of vector mortality, deterrence and blood-feeding inhibition, particularly when combined with LLINs. Two generations of commercial ITWL have been manufactured to date containing either pyrethroid or non-pyrethroid formulations. While some Phase III trials of these products have demonstrated reductions in malaria incidence, further large-scale evidence is still required before operational implementation of ITWL can be considered either in a programmatic or more targeted community context. Qualitative studies of ITWL have identified aesthetic value and observable entomological efficacy as key determinants of household acceptability. However, concerns have been raised regarding installation feasibility and anticipated cost-effectiveness. This paper critically reviews ITWL as both a putative mechanism of house improvement or more conventional intervention and discusses its future prospects as a method for controlling malaria and other vector-borne diseases.

Background Despite the availability of a highly effective vaccine, yellow fever virus (YFV) remains an important public health problem across Africa and South America due to its high case-fatality rate. This study investigated the historical epidemiology and contemporary entomological and social determinants of a YFV outbreak in South Omo Zone (SOZ), Ethiopia. Methods A YFV outbreak occurred in SOZ, Ethiopia in 2012–2014. Historical epidemiological data were retrieved from the SOZ Health Department and analyzed. Entomological sampling was undertaken in 2017, including mosquito species identification and molecular screening for arboviruses to understand mosquito habitat distribution, and finally current knowledge, attitudes and preventative practices within the affected communities were assessed. Results From October 2012 to March 2014, 165 suspected cases and 62 deaths were reported, principally in rural areas of South Ari region (83.6%). The majority of patients were 15–44 years old (75.8%) and most case deaths were males (76%). Between June and August 2017, 688 containers were sampled across 180 households to identify key breeding sites for Aedes mosquitoes. Ensete ventricosum (“false banana”) and clay pots outside the home were the most productive natural and artificial breeding sites, respectively. Entomological risk indices classified most sites as “high risk” for future outbreaks under current World Health Organization criteria. Adult mosquitoes in houses were identified as members of the Aedes simpsoni complex but no YFV or other arboviruses were detected by PCR. The majority of community members had heard of YFV, however few activities were undertaken to actively reduce mosquito breeding sites. Discussion Study results highlight the potential role vector control could play in mitigating local disease transmission and emphasize the urgent need to strengthen disease surveillance systems and in-country laboratory capacity to facilitate more rapid responses to future YFV outbreaks.

We report the characterization of Trypanosoma cruzi of southern South American origin among humans, domestic vectors, and peridomestic hosts in Colombia using high-resolution nuclear and mitochondrial genotyping. Expanding our understanding of the geographic range of lineage TcVI, which is associated with severe Chagas disease, will help clarify risk of human infection for improved disease control.

Abstract Background Despite considerable reductions in malaria achieved by scaling-up long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), maintaining sustained community protection remains operationally challenging. Increasing insecticide resistance also threatens to jeopardize the future of both strategies. Non-pyrethroid insecticide­treated wall lining (ITWL) may represent an alternate or complementary control method and a potential tool to manage insecticide resistance. To date no study has demonstrated whether ITWL can reduce malaria transmission nor provide additional protection beyond the current best practice of universal coverage (UC) of LLINs and prompt case management. Methods/design A two-arm cluster randomized controlled trial will be conducted in rural Tanzania to assess whether non-pyrethroid ITWL and UC of LLINs provide added protection against malaria infection in children, compared to UC of LLINs alone. Stratified randomization based on malaria prevalence will be used to select 22 village clusters per arm. All 44 clusters will receive LLINs and half will also have ITWL installed on interior house walls. Study children, aged 6 months to 11 years old, will be enrolled from each cluster and followed monthly to estimate cumulative incidence of malaria parasitaemia (primary endpoint), time to first malaria episode and prevalence of anaemia before and after intervention. Entomological inoculation rate will be estimated using indoor CDC light traps and outdoor tent traps followed by detection of Anopheles gambiae species, sporozoite infection, insecticide resistance and blood meal source. ITWL bioefficacy and durability will be monitored using WHO cone bioassays and household surveys, respectively. Social and cultural factors influencing community and household ITWL acceptability will be explored through focus-group discussions and in-depth interviews. Cost-effectiveness, compared between study arms, will be estimated per malaria case averted. Discussion This protocol describes the large-scale evaluation of a novel vector control product, designed to overcome some of the known limitations of existing methods. If ITWL is proven to be effective and durable under field conditions, it may warrant consideration for programmatic implementation, particularly in areas with long transmission seasons and where pyrethroid-resistant vectors predominate. Trial findings will provide crucial information for policy makers in Tanzania and other malaria-endemic countries to guide resource allocations for future control efforts. Trial registration NCT02533336 registered on 13 July 2014.

Abstract Background Trypanosoma cruzi, causative agent of Chagas disease, displays high intraspecific genetic diversity: six genetic lineages or discrete typing units (DTUs) are currently recognized, termed TcI through TcVI. Each DTU presents a particular distribution pattern across the Americas, and is loosely associated with different transmission cycles and hosts. Several DTUs are known to circulate in Central America. It has been previously suggested that TcI infection is benign and does not lead to chronic chagasic cardiomyopathy (CCC). Findings In this study, we genotyped T. cruzi parasites circulating in the blood and from explanted cardiac tissue of an El Salvadorian patient who developed reactivation Chagas disease while on immunosuppressive medications after undergoing heart transplant in the U.S. as treatment for end-stage CCC. Parasite typing was performed through molecular methods (restriction fragment length polymorphism of polymerase reaction chain amplified products, microsatellite typing, maxicircle sequence typing and low-stringency single primer PCR, [LSSP-PCR]) as well as lineage-specific serology. We show that the parasites infecting the patient belong to the TcI DTU exclusively. Our data indicate that the parasites isolated from the patient belong to a genotype frequently associated with human infection throughout the Americas (TcIDOM). Conclusions Our results constitute compelling evidence in support of TcI DTU’s ability to cause end-stage CCC and help dispel any residual bias that infection with this lineage is benign, pointing to the need for increased surveillance for dissemination of this genotype in endemic regions, the USA and globally.

Oral outbreaks of Chagas disease are increasingly reported in Latin America. The transitory presence of Trypanosoma cruzi parasites within contaminated foods, and the rapid consumption of those foods, precludes precise identification of outbreak origin. We report source attribution for 2 peri-urban oral outbreaks of Chagas disease in Venezuela via high resolution microsatellite typing.