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Abstract Background Spatial and longitudinal monitoring of transmission intensity will allow better targeting of malaria interventions. In this study, data on meteorological, demographic, entomological and parasitological data over the course of a year was collected to describe malaria epidemiology in a single village of low transmission intensity. Methods Entomological monitoring of malaria vectors was performed by weekly light trap catches in 10 houses. Each house in the village of Msitu wa Tembo, Lower Moshi, was mapped and censused. Malaria cases identified through passive case detection at the local health centre were mapped by residence using GIS software and the incidence of cases by season and distance to the main breeding site was calculated. Results The principle vector was Anopheles arabiensis and peak mosquito numbers followed peaks in recent rainfall. The entomological inoculation rate estimated was 3.4 (95% CI 0.7–9.9) infectious bites per person per year. The majority of malaria cases (85/130) occurred during the rainy season (χ2 = 62,3, p < 0.001). Living further away from the river (OR 0.96, CI 0.92–0.998, p = 0.04 every 50 m) and use of anti-insect window screens (OR 0.65, CI 0.44–0.94, p = 0.023) were independent protective factors for the risk of malaria infection. Children aged 1–5 years and 5–15 years were at greater risk of clinical episodes (OR 2.36, CI 1.41–3.97, p = 0.001 and OR 3.68, CI 2.42–5.61, p < 0.001 respectively). Conclusion These data show that local malaria transmission is restricted to the rainy season and strongly associated with proximity to the river. Transmission reducing interventions should, therefore, be timed before the rain-associated increase in mosquito numbers and target households located near the river.

The microbial larvicides Bacillus thuringiensis var. israelensis (Bti) and Bacillus sphaericus (Bs) (Bacillales: Bacillaceae) are well known for their efficacy and safety in mosquito control. In order to assess their potential value in future mosquito control strategies in western Kenya, the current study tested the susceptibility of five populations of Anopheles gambiae complex mosquitoes (Diptera: Culicidae), collected from five diverse ecological sites in this area, to Bti and Bs under laboratory conditions. In each population, bioassays were conducted with eight concentrations of larvicide (Bti/Bs) in four replicates and were repeated on three separate days. Larval mortality was recorded at 24 h or 48 h after the application of larvicide and subjected to probit analysis. A total of 2400 An. gambiae complex larvae from each population were tested for their susceptibility to Bti and Bs. The mean (± standard error of the mean, SEM) lethal concentration values of Bti required to achieve 50% and 95% larval mortality (LC50 and LC95 ) across the five populations were 0.062  (± 0.005) mg/L and 0.797 (± 0.087) mg/L, respectively. Corresponding mean  (± SEM) values for Bs were 0.058 (± 0.005) mg/L and 0.451 (± 0.053) mg/L, respectively. Statistical analysis indicated that the five populations of An. gambiae complex mosquitoes tested were fully susceptible to Bti and Bs, and there was no significant variation in susceptibility among the tested populations.

New classes of long-lasting insecticidal nets (LLINs), have been recommended by the World Health Organization (WHO) to control malaria vectors resistant to pyrethroid insecticides. This study was nested in a large-scale cluster-randomized controlled trial conducted (cRCT) in Tanzania. A series of experimental hut trials (EHTs) aimed to evaluate the bio-efficacy of trial LLINs on mosquito indicators most pertinent to malaria transmission over 3 years of use in the community in order to better understand the outcomes of the cRCT. The following field-collected LLINs were assessed: 1) Olyset Plus (combining piperonyl butoxide synergist and permethrin), 2) Interceptor G2 (chlorfenapyr and alpha-cypermethrin), 3) Royal Guard (pyriproxyfen and alpha-cypermethrin), 4) Interceptor (alpha-cypermethrin only) conducted in parallel with 5) a new Interceptor, and 6) an untreated net. Thirty nets of each type were withdrawn from the community at 12, 24, and 36 months after distribution and used for the EHTs. Pre-specified outcomes were 72-hour mortality for Interceptor G2, 24-hour mortality for Olyset Plus, and fertility based on egg development stage for Royal Guard. Overall, Interceptor G2 LLINs induced higher 72-hour mortality compared to standard LLINs of the same age up to12 months (44% vs 21%, OR: 3.5, 95% CI: 1.9-6.6, p-value < 0.001), and 24-hour mortality was only significantly higher in Olyset Plus when new (OR: 13.6, 95%CI: 4.4-41.3, p-value < 0.001) compared to standard LLINs but not at 12 months (17% vs 13%; OR: 2.1, 95% CI: 1.0-4.3; p-value = 0.112). A small, non-significant effect of pyriproxyfen on Anopheles fertility was observed for Royal Guard up to 12 months (75% vs 98%, OR: 1.1, 95% CI: 0.0-24.9, p-value = 0.951). There was no evidence of a difference in the main outcomes for any of the new classes of LLINs at 24 and 36 months compared to standard LLINs. Interceptor G2 LLINs showed superior bio-efficacy compared to standard LLINs only up to 12 months, and the effect of Olyset Plus was observed when new for all species and 12 months for An. gambiae s.l. only. The pyriproxyfen component of Royal Guard had a short and limited effect on fertility. The decrease in effectiveness of Olyset Plus and Royal Guard LLINs in the EHTs aligns with findings from the cRCT, whereas efficacy of Interceptor G2 lasted for a longer period in the cRCT compared to the EHT. Further investigations are needed to understand the complete scope of chlorfenapyr mode of action. Additional EHT in various contexts will help confirm the residual efficacy of the dual active ingredient LLINs and support the development of longer-lasting nets., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Next-generation insecticide-treated bed nets (ITNs) combining two insecticides or an insecticide with a synergist are vital in combating malaria, especially in areas with pyrethroid-resistant mosquitoes where standard pyrethroid long-lasting insecticidal net (LLIN) may be less effective. A community durability study was conducted in Misungwi, Tanzania, during a cluster randomised controlled trial. This study assessed the bio-efficacy of three net brands combining a pyrethroid insecticide and either a synergist PBO for Olyset Plus, or a second insecticide pyriproxyfen for Royal Guard, and chlorfenapyr for Interceptor G2 over three years. These nets were compared to Interceptor, a standard pyrethroid-only net. A total of 1950 nets were enrolled across 10 clusters in each treatment arm. Thirty nets per type were collected every 6 months up to 30 months, with 50 nets sampled at 36 months. WHO cone bioassays and tunnel tests were performed at 0, 12, 24, 30 and 36 months. Both susceptible An. gambiae ( s.s. ) Kisumu strain and resistant An. gambiae ( s.s. ) Muleba-Kis strain were exposed. Over 80% of nets tested against the susceptible Kisumu strain met the WHO criteria after three years of community use. In tunnel tests, mortality (72 h) of the resistant Anopheles varied between 52% and 20%, in Interceptor G2 and was higher than standard Interceptor net up to 24 months. Olyset Plus mortality (24 h) ranged between 84% and 33% in tunnel tests with superior efficacy compared to Interceptor at 0, 24 and 36 months. Sterility effects in Royal Guard were higher when these nets were new and at six months but decreased to less than 10% after 12 months. Royal Guard consistently induced higher mortality compared to Interceptor up to 30 months while next-generation ITNs demonstrated higher efficacy in terms of mortality compared to standard LLINs against resistant strains; this superior bio-efficacy did not persist for the full three years. The impact of active ingredient (dual-AI) and PBO diminished relatively quickly. Aside from the initial period when the nets were new, the differences in mortality for Interceptor G2 and Olyset Plus and in sterility for Royal Guard, compared to the standard LLINs, were relatively small thereafter., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors.)

Background: The Dual-Active Ingredient long-lasting insecticidal nets (Dual-AI LLIN) have been developed to counteract the reduced efficacy of pyrethroid (PY)-only nets due to widespread pyrethroid insecticide resistance in malaria vector mosquitoes. They constitute half of the nets distributed in sub-Saharan Africa between 2022 and 2024. However, their effectiveness once they develop holes is unclear, particularly in pyrethroid-resistant settings. This study evaluates the textile integrity of three dual- AI LLINs compared to standard PY LLN, over 3 years of use in a community in Tanzania and the associated impact on malaria prevalence and incidence., Methods: A secondary analysis of data from a randomized controlled trial (RCT) in North-western Tanzania was conducted to evaluate the effectiveness of α-cypermethrin only; pyriproxyfen and α-cypermethrin (PPF-PY); chlorfenapyr and α-cypermethrin (chlorfenapyr-PY); and the synergist piperonyl butoxide and permethrin (PBO-PY) LLINs on malaria infection prevalence and case incidence. The association between the net textile condition and 1/malaria prevalence over 3 years of use between 2019 and 2022, and 2/malaria case incidence in a cohort of children over 2 years of follow-up was assessed between 2019 and 2021., Results: There was no significant association between damaged (OR 0.98, 95% CI 0.71-1.37, p-value  = 0.655) and too-torn (OR 1.07, 95% CI 0.77-1.47, p-value = 0.694) compared to intact nets on malaria prevalence for all net types. However, there were reduced rates of malaria case incidence in children sleeping under a net in good condition compared to too-torn nets (incidence rate ratio (IRR) 0.76 [95% CI 0.63-0.92], p = 0.005). Malaria incidence was also consistently lower in too-torn PBO-PY LLIN (IRR = 0.37 [95% CI 0.19-0.72], p = 0.003) and chlorfenapyr-PY LLIN (IRR = 0.45 [95% CI 0.33-0.97], p = 0.053) compared to an intact PY-only LLIN during the first year of follow up. In year 2, the incidence was only significantly lower in intact chlorfenapyr-PY LLIN (IRR = 0.49 [95% CI 0.29-0.81], p = 0.006) compared to intact PY LLIN., Conclusion: The study confirmed that sleeping under a chlorfenapyr-PY LLIN or PBO-PY LLIN offered superior protection to pyrethroid-only nets even when torn. Preventing the development of holes is essential as they impact the level of protection offered against malaria infection., Trial Registration: ClinicalTrials.gov, number (NCT03554616)., (© 2024. The Author(s).)

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.

Background: New classes of long-lasting insecticidal nets (LLINs) containing two active ingredients have been recently recommended by WHO in areas where malaria vectors are resistant to pyrethroids. This policy was based on evidence generated by the first 2 years of our recently published trial in Tanzania. In this Article, we report the final third-year trial findings, which are necessary for assessing the long-term effectiveness of new classes of LLIN in the community and the replacement intervals required., Methods: A third year of follow-up of a four-arm, single-blind, cluster-randomised controlled trial of dual active ingredient LLINs was conducted between July 14, 2021, and Feb 10, 2022, in Misungwi, Tanzania. Restricted randomisation was used to assign 84 clusters to the four LLIN groups (1:1:1:1) to receive either standard pyrethroid (PY) LLINs (reference), chlorfenapyr-PY LLINs, pyriproxyfen-PY LLINs, or piperonyl butoxide (PBO)-PY LLINs. All households received one LLIN for every two people. Data collection was done in consenting households in the cluster core area with at least one child between 6 months and 15 years of age who permanently resided in the selected household. Exclusion criteria were householders absent during the visit, living in the cluster buffer area, no adult caregiver capable of giving informed consent, or eligible children who were severely ill. Field staff and study participants were masked to allocation, and those analysing data were not. The primary 24-month endpoint was reported previously; here, we present the secondary outcome, malaria infection prevalence in children at 36 months post LLIN distribution, reported in the intention-to-treat analysis. The trial was registered with ClinicalTrials.gov (NCT03554616) and is now complete., Findings: Overall usage of study nets was 1023 (22·3%) of 4587 people at 36 months post distribution. In the standard PY LLIN group, malaria infection was prevalent in 407 (37·4%) of 1088 participants, compared with 261 (22·8%) of 1145 in the chlorfenapyr-PY LLIN group (odds ratio 0·57, 95% CI 0·38-0·86; p=0·0069), 338 (32·2%) of 1048 in the PBO-PY LLIN group (0·95, 0·64-1·42; p=0·80), and 302 (28·8%) of 1050 in the pyriproxyfen-PY LLIN group (0·82, 0·55-1·23; p=0·34). None of the participants or caregivers reported side-effects., Interpretation: Despite low coverage, the protective efficacy against malaria offered by chlorfenapyr-PY LLINs was superior to that provided by standard PY LLINs over a 3-year LLIN lifespan. Appropriate LLIN replacement strategies to maintain adequate usage of nets will be necessary to maximise the full potential of these nets., Funding: Department for International Development, UK Medical Research Council, Wellcome Trust, Department of Health and Social Care, and Bill & Melinda Gates Foundation via the Innovative Vector Control Consortium., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Background: After a decade of successful control, malaria is on the rise again. The prevalence of malaria in Tanzania has increased from 7% in 2017 to 8% in 2022 and reached 18% in Kagera region in the North West of Tanzania. Malaria vectors in Muleba district Kagera have high level of pyrethroid resistance. The aim of this paper is to explore factors associated with malaria infection prevalence in children aged 6 months to 14 years in Muleba, where Long Lasting Insecticidal Net (LLIN) combining a pyrethroid insecticide and synergist piperonyl butoxide (PBO) that counteract resistance in the mosquitoes, was first distributed under trial conditions in 2015., Methods: The trial was a community randomized control in which there were two malaria prevalence cross-sectional household surveys each year (June and December) from 2015 to 2017 in Muleba. In this study we conducted a secondary data analysis of the December surveys only. Multilevel Poisson regression analysis was used to assess factors associated with malaria infection., Results: A total of 10,941 children and 4,611 households were included in this study. Overall malaria prevalence was 35.8%, 53.3% and 54.4% in the year 2015, 2016 and 2017 respectively. Living in an area with standard LLIN as opposed to the novel PBO synergist LLIN, being a male child, above 5 years of age, living in a house with open eaves, living in house without IRS, having head of household with no formal education, lower socioeconomic status and survey year were associated with increased risk of malaria infection., Conclusions: Using PBO LLIN reduced the risk of malaria infection. However, additional measures could further reduce malaria infection in areas of insecticide resistance such as housing improvement., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kibondo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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)., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Background: Insecticide resistance among malaria-vector species is a pervasive problem that might jeopardise global disease-control efforts. Novel vector-control tools with different modes of action, including long-lasting insecticidal nets (LLINs) incorporating new active ingredients, are urgently needed to delay the evolution and spread of insecticide resistance. We aimed to measure phenotypic and genotypic insecticide-resistance profiles among wild Anopheles collected over 3 years to assess the longitudinal effects of dual-active-ingredient LLINs on insecticide resistance., Methods: For this analysis, data nested in a 3-year, four parallel-arm, superiority cluster-randomised controlled trial (cRCT) in Tanzania, collected from 84 clusters (39 307 households) formed of 72 villages in the Misungwi district, were used to measure insecticide-resistance profiles among female Anopheles mosquitoes via insecticide-resistance bioassays and quantitative RT-PCR of metabolic-resistance genes. Wild, blood-fed, indoor-resting mosquitoes were collected annually during the rainy seasons from house walls in clusters from all four trial groups. Mosquitoes were morphologically identified as An gambiae sensu lato (SL) or An funestus SL before separate bioassay testing. The primary outcomes were lethal-dose values for α-cypermethrin, permethrin, and piperonyl butoxide pre-exposure plus permethrin-resistance intensity bioassays, mortality 72 h after insecticidal exposure for chlorfenapyr bioassays, fertility reduction 72 h after insecticidal exposure for pyriproxyfen bioassays, and fold change in metabolic-enzyme expression relative to an insecticide-susceptible laboratory strain. All primary outcomes were measured in An funestus SL 1 year, 2 years, and 3 years after LLIN distribution. Primary outcomes were also assessed in An gambiae SL if enough mosquitoes were collected. The cRCT is registered with ClinicalTrials.gov (NCT03554616)., Findings: Between May 24, 2019, and Oct 25, 2021, 47 224 female Anopheles were collected for resistance monitoring. In the pyrethroid (PY)-LLIN group, there were significant increases in α-cypermethrin-resistance intensity (year 1 LD50=9·52 vs year 2 76·20, p<0·0001) and permethrin-resistance intensity (year 1 13·27 vs year 2 35·83, p=0·0019) in An funestus SL. In the pyriproxyfen PY-LLIN group, there was similar increase in α-cypermethrin-resistance intensity (year 1 0·71 vs year 2 81·56, p<0·0001) and permethrin-resistance intensity (year 1 5·68 vs year 2 50·14, p<0·0001). In the piperonyl butoxide PY-LLIN group, α-cypermethrin-resistance intensity (year 1 33·26 vs year 3 70·22, p=0·0071) and permethrin-resistance intensity (year 1 47·09 vs year 3 2635·29, p<0·0001) also increased over time. In the chlorfenapyr PY-LLIN group, there were no effects on α-cypermethrin-resistance intensity (year 1 0·42 vs year 3 0·99, p=0·54) or permethrin-resistance intensity (data were not estimable due to nearly 100% mortality). There were also minimal reductions in chlorfenapyr susceptibility. However, in the chlorfenapyr PY-LLIN group, a significant decline in piperonyl-butoxide synergy was seen by year 3 (year 1 0·02 vs year 3 0·26, p=0·020). Highly over-expressed detoxification enzymes showed dynamic patterns of selection throughout the trial., Interpretation: Our phenotypic data supports trial epidemiological findings; chlorfenapyr PY-LLINs provided superior protection from malaria across multiple transmission seasons, with few effects on insecticide-resistance selection. Rapid pyrethroid-resistance intensification in the piperonyl butoxide PY-LLIN group and pre-existing tolerance of pyriproxyfen in vector populations might explain the poorer performance of these two interventions regarding malaria outcomes. Further work is required to elucidate the potential mechanisms driving cross-resistance between pyrethroids and novel active ingredients to better inform the design of pre-emptive resistance-management strategies., Funding: UK Department for International Development; UK Medical Research Council; Wellcome Trust; UK Department of Health and Social Care; UK Foreign, Commonwealth and Development Office; and The Bill and Melinda Gates Foundation via the Innovative Vector Control Consortium., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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.

Background: Gains in malaria control are threatened by widespread pyrethroid resistance in malaria vectors across sub-Saharan Africa. New long-lasting insecticidal nets (LLINs) containing two active ingredients (dual active-ingredient LLINs) have been developed to interrupt transmission in areas of pyrethroid resistance. We aimed to evaluate the effectiveness of three dual active-ingredient LLINs compared with standard pyrethroid LLINs against pyrethroid-resistant malaria vectors in rural Tanzania., Methods: In this study, we did a secondary analysis of entomological data from a four-group, 3 year, single-blind, cluster-randomised controlled trial carried out between Feb 18, 2019, and Dec 6, 2021. We conducted quarterly indoor mosquito collections using the Centers for Disease Control and Prevention light trap, in eight houses in each of the 84 study clusters in the Misungwi district, northwestern Tanzania. Anopheles vectors were then tested for malaria parasites and identified at species level, to distinguish between sibling species of the Anopheles gambiae and Anopheles funestus groups, using molecular laboratory techniques. The primary outcomes were density of different malaria vector species measured as the number of female Anopheles collected per household per night, the entomological inoculation rate (EIR), an indicator of malaria transmission, and sporozoite rate. Entomological outcomes were assessed on the basis of intention to treat, and the effect of the three dual active-ingredient LLINs was compared with the standard pyrethroid LLINs at household level., Findings: Dual active-ingredient LLINs had the greatest effect on Anopheles funestus sl, the most efficient vector in the study area, with comparatively weak effect on An arabiensis. An funestus density was 3∙1 per house per night in the pyrethroid LLIN group, 1∙2 in the chlorfenapyr pyrethroid LLIN group (adjusted density ratio [aDR]=0∙26, 95% CI 0∙17-0∙14, p<0∙0001), 1∙4 in the piperonyl-butoxide pyrethroid LLIN group (aDR=0∙49, 0∙32-0∙76, p=0∙0012), and 3∙0 in the pyriproxyfen pyrethroid LLIN group (aDR=0∙72, 0∙47-1∙11, p=0∙15). Malaria transmission intensity was also significantly lower in the chlorfenapyr pyrethroid group, with 0∙01 versus 0∙06 infective bites per household per night in the pyrethroid LLIN group (aDR=0∙21, 0∙14-0∙33, p<0∙0001). Ecological niche models indicated that vector-species distribution was stable following LLIN intervention despite the reductions observed in An funestus sl density., Interpretation: Chlorfenapyr pyrethroid LLINs were the most effective intervention against the main malaria vector An funestus sl over 3 years of community use, whereas the effect of piperonyl-butoxide pyrethroid LLIN was sustained for 2 years. The other vector, An arabiensis, was not controlled by any of the dual active-ingredient LLINs. Additional vector control tools and strategies targeted to locally prevalent vector species evading dual active-ingredient LLINs should be deployed to further reduce malaria transmission and achieve elimination., Funding: The Department for International Development, UK Medical Research Council, Wellcome Trust, the Department of Health and Social Care, and The Bill & Melinda Gates Foundation via the Innovative Vector Control Consortium., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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 LC 99  × 2 = 1.126-54.00 μg/ml or LC 95  × 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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Lukole et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Optimising insecticide use and managing insecticide resistance are important to sustain gains against malaria using long-lasting insecticidal nets (LLINs). Restricting insecticides to where mosquitoes are most likely to make multiple contacts could reduce the quantity of insecticide needed to treat the nets. Previous studies have shown that nets partially treated with a pyrethroid insecticide had equivalent mortality compared to a fully treated net. This study compared the efficacy of: (i) whole Interceptor® G2 nets (IG2; a dual-active LLIN containing alpha-cypermethrin and chlorfenapyr), (ii) nets with roof panels made of IG2 netting, (iii) nets with side panels made of IG2 netting and (iv) whole untreated nets as test nets., Methods: The study was conducted in cow-baited experimental huts, Moshi Tanzania, using a four-arm Latin square design. Test nets had 30 holes cut in panels to simulate a typical net after 2-3 year use. The trial data were analysed using generalized linear models with mortality, blood-feeding, exophily and deterrence against wild free-flying Anopheles arabiensis as outcomes and test nets as predictors., Results: Mortality was significantly higher in the nets with roof IG2 [27%, P = 0.001, odds ratio (OR) = 51.0, 95% CI = 4.8-546.2), side IG2 (44%, P < 0.001, OR = 137.6, 95% CI = 12.2-1553.2] and whole IG2 (53%, P < 0.001, OR = 223.0, 95% CI = 19.07-2606.0) nettings than the untreated (1%) nets. Mortality was also significantly higher in the whole IG2 net compared to the net with roof IG2 netting (P = 0.009, OR = 4.4, 95% CI = 1.4-13.3). Blood feeding was 22% in untreated, 10% in roof IG2, 14% in side IG2 and 19% in whole IG2 nets. Exiting was 92% in untreated, 89% in roof IG2, 97% in side IG2 and 94% whole IG2 nets., Conclusion: The results show that although the roof-treated IG2 net induced greater mortality compared to untreated nets, its efficacy was reduced compared to whole IG2 nets. Therefore, there was no benefit to be gained from restricting dual-active ingredient IG2 netting to the roof of nets., (© 2022. The Author(s).)

Malaria rapid diagnosis test (RDT) is crucial for managing the disease, and the effectiveness of detection depends on parameters such as sensitivity and specificity of the RDT. Several factors can affect the performance of RDT. In this study, we focused on the pfhrp2 sequence variation and its impact on RDTs targeted by antigens encoded by Plasmodium falciparum histidine-rich protein 2 ( pfhrp2) . Field samples collected during cross-sectional surveys in Tanzania were sequenced to investigate the pfhrp2 sequence diversity and evaluate the impact on HRP2-based RDT performance. We observed significant mean differences in amino acid repeats between current and previous studies. Several new amino acid repeats were found to occur at different frequencies, including types AAY, AHHAHHAAN, and AHHAA. Based on the abundance of types 2 and 7 amino acid repeats, the binary predictive model was able to predict RDT insensitivity by about 69% in the study area. About 85% of the major epitopes targeted by monoclonal antibodies (MAbs) in RDT were identified. Our study suggested that the extensive sequence variation in pfhrp2 can contribute to reduced RDT sensitivity. The correlation between the different combinations of amino acid repeats and the performance of RDT in different malaria transmission settings should be investigated further.

Indoor residual spraying (IRS) has changed little since its introduction in the 1940s. Manual spraying is still prone to variation in insecticide dose. To improve the application of IRS in experimental hut trials, an automated track sprayer was developed, which regulates the speed of application and the distance of the nozzle from the wall, two key sources of variation. The automated track sprayer was compared to manual spraying, firstly using fluorescein solution in controlled indoor settings, and secondly in experimental huts in Tanzania using several IRS products. Manual spraying produced greater variation with both fluorescein and insecticide applications. Both manual and automated spray methods under-dosed the actual dose sprayed compared to the target dose. Overall, the track sprayer treats surfaces more consistently, offering a potential improvement over manual spraying for experimental hut evaluation of new IRS formulations.

Plasmodium falciparum parasites lacking histidine-rich protein 2 and 3 (pfhrp2/3) genes have been reported in several parts of the world. These deletions are known to compromise the effectiveness of HRP2-based malaria rapid diagnostic tests (HRP2-RDT). The National Malaria Control Programme (NMCP) in Tanzania adopted HRP2-RDTs as a routine tool for malaria diagnosis in 2009 replacing microscopy in many Health facilities. We investigated pfhrp2/3 deletions in 122 samples from two areas with diverse malaria transmission intensities in Northeastern Tanzania. Pfhrp2 deletion was confirmed in 1.6% of samples while pfhrp3 deletion was confirmed in 50% of samples. We did not find parasites with both pfhrp2 and pfhrp3 deletions among our samples. Results from this study highlight the need for systematic surveillance of pfhrp2/3 deletions in Tanzania to understand their prevalence and determine their impact on the performance of mRDT., (© 2022. The Author(s).)

Background: Long-lasting insecticidal nets (LLINs) have successfully reduced malaria in sub-Saharan Africa, but their effectiveness is now partly compromised by widespread resistance to insecticides among vectors. We evaluated new classes of LLINs with two active ingredients with differing modes of action against resistant malaria vectors., Methods: We did a four-arm, cluster-randomised trial in Misungwi, Tanzania. Clusters were villages, or groups of hamlets, with at least 119 households containing children aged 6 months to 14 years living in the cluster's core area. Constrained randomisation was used to allocate clusters (1:1:1:1) to receive one of four types of LLIN treated with the following: α-cypermethrin only (pyrethroid-only [reference] group); pyriproxyfen and α-cypermethrin (pyriproxyfen group); chlorfenapyr and α-cypermethrin (chlorfenapyr group); or the synergist piperonyl butoxide and permethrin (piperonyl butoxide group). At least one LLIN was distributed for every two people. Community members and the field team were masked to group allocation. Malaria prevalence data were collected through cross-sectional surveys of randomly selected households from each cluster, in which children aged 6 months to 14 years were assessed for Plasmodium falciparum malaria infection by rapid diagnostic tests. The primary outcome was malaria infection prevalence at 24 months after LLIN distribution, comparing each of the dual-active-ingredient LLINs to the standard pyrethroid-only LLINs in the intention-to-treat population. The primary economic outcome was cost-effectiveness of dual-active-ingredient LLINs, based on incremental cost per disability-adjusted life-year (DALY) averted compared with pyrethroid-only LLINs, modelled over a 2-year period; we included costs of net procurement and malaria diagnosis and treatment, and estimated DALYs in all age groups. This study is registered with ClinicalTrials.gov (NCT03554616), and is ongoing but no longer recruiting., Findings: 84 clusters comprising 39 307 households were included in the study between May 11 and July 2, 2018. 147 230 LLINs were distributed among households between Jan 26 and Jan 28, 2019. Use of study LLINs was reported in 3155 (72·1%) of 4378 participants surveyed at 3 months post-distribution and decreased to 8694 (40·9%) of 21 246 at 24 months, with varying rates of decline between groups. Malaria infection prevalence at 24 months was 549 (45·8%) of 1199 children in the pyrethroid-only reference group, 472 (37·5%) of 1258 in the pyriproxyfen group (adjusted odds ratio 0·79 [95% CI 0·54-1·17], p=0·2354), 512 (40·7%) of 1259 in the piperonyl butoxide group (0·99 [0·67-1·45], p=0·9607), and 326 [25·6%] of 1272 in the chlorfenapyr group (0·45 [0·30-0·67], p=0·0001). Skin irritation or paraesthesia was the most commonly reported side-effect in all groups. Chlorfenapyr LLINs were the most cost-effective LLINs, costing only US$19 (95% uncertainty interval 1-105) more to public providers or $28 (11-120) more to donors per DALY averted over a 2-year period compared with pyrethroid-only LLINs, and saving costs from societal and household perspectives., Interpretation: After 2 years, chlorfenapyr LLINs provided significantly better protection than pyrethroid-only LLINs against malaria in an area with pyrethroid-resistant mosquitoes, and the additional cost of these nets would be considerably below plausible cost-effectiveness thresholds ($292-393 per DALY averted). Before scale-up of chlorfenapyr LLINs, resistance management strategies are needed to preserve their effectiveness. Poor textile and active ingredient durability in the piperonyl butoxide and pyriproxyfen LLINs might have contributed to their relative lack of effectiveness compared with standard LLINs., Funding: Joint Global Health Trials scheme (UK Foreign, Commonwealth and Development Office; UK Medical Research Council; Wellcome; UK Department of Health and Social Care), US Agency for International Development, President's Malaria Initiative., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

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., (© 2022. The Author(s).)

Objective: A community-based cross-sectional study was done to assess Plasmodium falciparum exposure in areas with different malaria endemicity in north-eastern Tanzania using serological markers; PfAMA-1 and PfMSP-1 19 ., Results: Bondo had a higher seroprevalence 36.6% (188) for PfAMA-1 as compared to Hai 13.8% (33), χ 2  = 34.66, p < 0.01. Likewise, Bondo had a higher seroprevalence 201(36.6%) for PfMSP-1 as compared to Hai 41 (17.2%), χ 2  = 29.62, p < 0.01. Anti-PfAMA-1 titters were higher in malaria positive individuals (n = 47) than in malaria negative individuals (n = 741) (p = 0.07). Anti-PfMSP-1 antibody concentrations were significantly higher in malaria-positive individuals (n = 47) than in malaria-negative individuals (n = 741) (p = 0.003). Antibody response against PfAMA-1 was significantly different between the three age groups; < 5 years, 5 to 15 years and > 15 years in both sites of Bondo and Hai. Likewise, antibody response against PfMSP-1 19 was significantly different between the three age groups in the two sites (p < 0.001). We also found significant differences in the anti-PfAMA-1and anti-PfMSP-1 19 antibody concentrations among the three age groups in the two sites (p = 0.004 and 0.005) respectively. Immunological indicators of P. falciparum exposure have proven to be useful in explaining long-term changes in the transmission dynamics, especially in low transmission settings., (© 2021. The Author(s).)

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.

Background: The spread of pyrethroid resistance in malaria vectors threatens the effectiveness of standard long-lasting insecticidal nets (LLIN). Synergist nets combine pyrethroid (Py) and piperonyl-butoxide (PBO) to enhance potency against resistance mediated by mono-oxygenase mechanisms. Our project assessed personal protection of the World Health Organization first-in-class PBO-Py LLIN (Olyset Plus) versus the standard LLIN (Olyset net) against pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) and An. funestus in North-West Tanzania after 20 months of household use., Methods: From a household survey, 39 standard Olyset net and 39 Olyset Plus houses were selected. The physical integrity and hole index (HI) of the nets were assessed, and resting mosquitoes were collected from inside nets and from room walls. The indoor abundance was estimated using CDC light traps and species identified using PCR. The bioefficacy of PBO and standard LLINs against wild Anopheles was assessed using 30-minute cylinder bioassays., Results: Of 2397 Anopheles collected, 8.9% (n = 213) were resting inside standard Olyset nets, while none were found inside Olyset Plus nets (PBO-Py LLINs) of any HI category. Resting density of blood-fed mosquitoes was higher on walls of sleeping rooms with Olyset nets compared to Olyset Plus (0.62 vs 0.10, density ratio [DR]: 0.03, 95% CI 0.01-0.13, p < 0.001). Mosquitoes were found inside Olyset nets of all WHO HI categories, but more were collected inside the more damaged nets (HI ≥ 643) than in less damaged (HI 0-64) nets (DR: 6.4, 95% CI 1.1-36.0, p = 0.037). In bioassay, mortality of An. gambiae s.l. was higher with Olyset Plus than with Olyset nets for new nets (76.8% vs 27.5%) and nets used for 20 months (56.8% vs 12.8%); similar trends were observed with An. funestus., Conclusion: The PBO-Py LLINs provided improved protection after 20 months of household use, as demonstrated by the higher bioassay mortality and absence of pyrethroid-resistant An. gambiae sensu stricto (s.s.) and An. funestus collected from inside Olyset Plus nets, irrespective of HI category, as compared to Olyset nets.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY. Published by BMJ.)

Novel chemistry for vector control is urgently needed to counter insecticide resistance in mosquitoes. Here a new meta-diamide insecticide, broflanilide (TENEBENALTM), was evaluated in East African experimental huts in Moshi, northern Tanzania. Two consecutive experimental hut trials with broflanilide 50WP were conducted; the first evaluating the efficacy of three concentrations, 50 mg/m2, 100 mg/m2, and 200 mg/m2 using a prototype formulation, and the second trial evaluating an improved formulation. The IRS treatments were applied on both mud and concrete surfaces and efficacy was monitored over time. The mortality, blood-feeding inhibition and exiting behaviour of free-flying wild mosquitoes was compared between treatment arms. Additionally, cone assays with pyrethroid-susceptible and resistant mosquito strains were conducted in the huts to determine residual efficacy. The first trial showed a dosage-mortality response of the prototype formulation and 3-8 months of residual activity, with longer activity on concrete than mud. The second trial with an improved formulation showed prolonged residual efficacy of the 100 mg/m2 concentration to 5-6 months on mud, and mosquito mortality on the concrete surface ranged between 94-100% for the full duration of the trial. In both trials, results with free-flying, wild Anopheles arabiensis echoed the mortality trend shown in cone assays, with the highest dose inducing the highest mortality and the improved formulation showing increased mortality rates. No blood-feeding inhibition or insecticide-induced exiting effects were observed with broflanilide. Broflanilide 50WP was effective against both susceptible and pyrethroid-resistant mosquito strains, demonstrating an absence of cross resistance between broflanilide and pyrethroids. The improved formulation, which has now been branded VECTRONTM T500, resulted in a prolonged residual efficacy. These results indicate the potential of this insecticide as an addition to the arsenal of IRS products needed to maintain both control of malaria and resistance management of malaria-transmitting mosquitoes., Competing Interests: The authors have declared that no competing interests exist.

Background: Long-lasting insecticidal nets (LLINs) are the most widely deployed vector control intervention in sub-Saharan Africa to prevent malaria. Recent reports indicate selection of pyrethroid insecticide resistance is widespread in mosquito vectors. This paper explores risk factors associated with malaria infection prevalence and vector density between mass distribution campaigns, changes in net coverage, and loss of protection in an area of high pyrethroid resistance in Northwest Tanzania., Methods: A cross sectional malaria survey of 3456 children was undertaken in 2014 in Muleba district, Kagera region west of Lake Victoria. Vector density was assessed using indoor light traps and outdoor tent traps. Anophelines were identified to species using PCR and tested for Plasmodium falciparum circumsporozoite protein. Logistic regression was used to identify household and environmental factors associated with malaria infection and regression binomial negative for vector density., Results: LLIN use was 27.7%. Only 16.9% of households had sufficient nets to cover all sleeping places. Malaria infection was independently associated with access to LLINs (OR: 0.57; 95% CI 0.34-0.98). LLINs less than 2 years old were slightly more protective than older LLINs (53 vs 65% prevalence of infection); however, there was no evidence that LLINs in good condition (hole index < 65) were more protective than LLINs, which were more holed. Other risk factors for malaria infection were age, group, altitude and house construction quality. Independent risk factors for vector density were consistent with malaria outcomes and included altitude, wind, livestock, house quality, open eaves and LLIN usage. Indoor collections comprised 4.6% Anopheles funestus and 95.4% Anopheles gambiae of which 4.5% were Anopheles arabiensis and 93.5% were Anopheles gambiae sensu stricto., Conclusion: Three years after the mass distribution campaign and despite top-ups, LLIN usage had declined considerably. While children living in households with access to LLINs were at lower risk of malaria, infection prevalence remained high even among users of LLINs in good condition. While effort should be made to maintain high coverage between campaigns, distribution of standard pyrethroid-only LLINs appears insufficient to prevent malaria transmission in this area of intense pyrethroid resistance.

Several trials and reviews have outlined the potential role of larviciding for malaria control in sub-Saharan Africa (SSA) to supplement the core indoor insecticide-based interventions. It has been argued that widespread use of long-lasting insecticide-treated nets (LLINs) and indoor residual spraying (IRS) interventions in many parts of Africa result in many new areas with low and focal malaria transmission that can be targeted with larvicides. As some countries in SSA are making good progress in malaria control, larval source management, particularly with bacterial larvicides, could be included in the list of viable options to maintain the gains achieved while paving the way to malaria elimination. We conducted a review of published literature that investigated the application of bacterial larvicides, Bacillus thuringiensis var. israelensis (Bti) and/or Bacillus sphaericus (Bs) for malaria vector control in SSA. Data for the review were identified through PubMed, the extensive files of the authors and reference lists of relevant articles retrieved. A total of 56 relevant studies were identified and included in the review. The findings indicated that, at low application rates, bacterial larvicide products based on Bti and/or Bs were effective in controlling malaria vectors. The larvicide interventions were found to be feasible, accepted by the general community, safe to the non-target organisms and the costs compared fairly well with those of other vector control measures practiced in SSA. Our review suggests that larviciding should gain more ground as a tool for integrated malaria vector control due to the decline in malaria which creates more appropriate conditions for the intervention and to the recognition of limitations of insecticide-based vector control tools. The advancement of new technology for mapping landscapes and environments could moreover facilitate identification and targeting of the numerous larval habitats preferred by the African malaria vectors. To build sustainable anti-larval measures in SSA, there is a great need to build capacity in relevant specialties and develop organizational structures for governance and management of larval source management programmes.

The microbial larvicides Bacillus thuringiensis var. israelensis and Bacillus sphaericus have been used extensively for mosquito control and have been found to be effective and safe to non-target organisms cohabiting with mosquito larvae. Recently developed long lasting microbial larvicides (LLML), although evading the previous challenge of short duration of activity, increase the risk of persistence of toxins in the treated larval habitats. This study monitored the impact of LLML FourStar ® and LL3 on non-target organisms cohabiting with mosquito larvae in an operational study to control malaria vectors in western Kenya highlands. A total of 300 larval habitats were selected in three highland villages. The habitats were first monitored for 5 weeks to collect baseline data on non-target organisms cohabiting with mosquito larvae and then randomized into two treatment arms (respective FourStar ® and LL3) and one control arm. Non-target organisms were sampled weekly for 5 months after treatment to assess the impact of LLML intervention. Before treatment, the mean density of all non-target organisms combined in the control, LL3 and FourStar ® treated habitats was 1.42, 1.39 and 1.49 individuals per habitat per sampling occasion, respectively. Following treatment, this density remained fairly unchanged for 21 weeks at which time it was 1.82, 2.11, and 2.05 for the respective control, LL3 and FourStar ® treated habitats. Statistical analysis revealed that LL3 and FourStar ® did not significantly alter abundance, richness or diversity of the 11 taxa studied, when comparing the intervention and control larval habitats. However, both FourStar ® and LL3 significantly reduced the density of malaria vectors. In conclusion, one round of label rate application of FourStar ® or LL3 in natural larval habitats did not alter richness, abundance or diversity of the monitored aquatic non-target organisms cohabiting with mosquito larvae to an ecologically significant level.

Background: Progress in malaria control is under threat by wide-scale insecticide resistance in malaria vectors. Two recent vector control products have been developed: a long-lasting insecticidal net that incorporates a synergist piperonyl butoxide (PBO) and a long-lasting indoor residual spraying formulation of the insecticide pirimiphos-methyl. We evaluated the effectiveness of PBO long-lasting insecticidal nets versus standard long-lasting insecticidal nets as single interventions and in combination with the indoor residual spraying of pirimiphos-methyl., Methods: We did a four-group cluster randomised controlled trial using a two-by-two factorial design of 48 clusters derived from 40 villages in Muleba (Kagera, Tanzania). We randomly assigned these clusters using restricted randomisation to four groups: standard long-lasting insecticidal nets, PBO long-lasting insecticidal nets, standard long-lasting insecticidal nets plus indoor residual spraying, or PBO long-lasting insecticidal nets plus indoor residual spraying. Both standard and PBO nets were distributed in 2015. Indoor residual spraying was applied only once in 2015. We masked the inhabitants of each cluster to the type of nets received, as well as field staff who took blood samples. Neither the investigators nor the participants were masked to indoor residual spraying. The primary outcome was the prevalence of malaria infection in children aged 6 months to 14 years assessed by cross-sectional surveys at 4, 9, 16, and 21 months after intervention. The endpoint for assessment of indoor residual spraying was 9 months and PBO long-lasting insecticidal nets was 21 months. This trial is registered with ClinicalTrials.gov, number NCT02288637., Findings: 7184 (68·0%) of 10 560 households were selected for post-intervention survey, and 15 469 (89·0%) of 17 377 eligible children from the four surveys were included in the intention-to-treat analysis. Of the 878 households visited in the two indoor residual spraying groups, 827 (94%) had been sprayed. Reported use of long-lasting insecticidal nets, across all groups, was 15 341 (77·3%) of 19 852 residents after 1 year, decreasing to 12 503 (59·2%) of 21 105 in the second year. Malaria infection prevalence after 9 months was lower in the two groups that received PBO long-lasting insecticidal nets than in the two groups that received standard long-lasting insecticidal nets (531 [29%] of 1852 children vs 767 [42%] of 1809; odds ratio [OR] 0·37, 95% CI 0·21-0·65; p=0·0011). At the same timepoint, malaria prevalence in the two groups that received indoor residual spraying was lower than in groups that did not receive indoor residual spraying (508 [28%] of 1846 children vs 790 [44%] of 1815; OR 0·33, 95% CI 0·19-0·55; p<0·0001) and there was evidence of an interaction between PBO long-lasting insecticidal nets and indoor residual spraying (OR 2·43, 95% CI 1·19-4·97; p=0·0158), indicating redundancy when combined. The PBO long-lasting insecticidal net effect was sustained after 21 months with a lower malaria prevalence than the standard long-lasting insecticidal net (865 [45%] of 1930 children vs 1255 [62%] of 2034; OR 0·40, 0·20-0·81; p=0·0122)., Interpretation: The PBO long-lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent and either intervention could be deployed to good effect. As a result, WHO has since recommended to increase coverage of PBO long-lasting insecticidal nets. Combining indoor residual spraying with pirimiphos-methyl and PBO long-lasting insecticidal nets provided no additional benefit compared with PBO long-lasting insecticidal nets alone or standard long-lasting insecticidal nets plus indoor residual spraying., Funding: UK Department for International Development, Medical Research Council, and Wellcome Trust., (Copyright © 2018 The Author(s). Published by Elsevier Ltd. This is an Open Access article under the CC BY 4.0 license. Published by Elsevier Ltd.. All rights reserved.)

Objective: Malaria vectors control interventions are designed to cause immediate killing or shorten mosquito lives, therefore does not allow enough time for the development of the parasites to infective stage. The wall lining is new malaria vectors control intervention in Tanzania where its impact on age structure is not well known. Therefore this study aimed at determining the impact of non-pyrethroid durable wall lining on the age structure of malaria vectors., Results: Higher proportions of An. gambiae sensu lato (57.1%, z = 2.66, P = 0.0077) and An. funestus (64.8%, z = 3.38, P = 0.001) were collected in the control clusters. Unexpectedly, significantly higher proportion of parous An. gambiae s. l. were collected in the intervention clusters (z = - 2.78, P = 0.0054). The wall lining intervention has demonstrated low impact on age structure of An. gambiae s. l., this call for further studies on the efficacy of the intervention.

Background: Mosquito nets containing synergists designed to overcome metabolic resistance mechanisms in vectors have been developed. These may enhance excitability in the mosquitoes and affect how they respond to CDC light-traps. Investigating the behaviour of vectors of disease in relation to novel mosquito nets is, therefore, essential for the design of sampling and surveillance systems., Methods: In an initial experiment in Muleba, Tanzania, nine bedrooms from three housing clusters were sampled. CDC light-traps were operated indoors next to occupied untreated nets (UTN), Olyset ® long lasting insecticidal net (LLIN) and Olyset Plus ® LLIN containing piperonyl butoxide (PBO) synergist. Nets were rotated daily between the nine rooms over nine nights. A further series of experiments using the nets on alternate nights in a single room was undertaken during the short rains. Anopheles gambiae s.l. were collected in CDC light-traps, a window-trap and Furvela tent-trap. Anopheles gambiae s.l. were identified to species by polymerase chain reaction (PCR)., Results: In the initial experiment 97.7% of the 310 An. gambiae s.l. were An. gambiae s.s., the remainder being Anopheles arabiensis. The number of mosquitoes collected from 81 light-trap collections was greater in the presence of an Olyset [density rate ratio 1.81, 95% CI (1.22-2.67), p = 0.003] relative to an UTN. In a second experiment, in the wet season 84% of the 180 An. gambiae s.l. identified were An. arabiensis. The number of An. gambiae s.l. collected from a light-trap compared to a tent-trap was significantly higher when an Olyset Plus net was used compared to an UTN. Survival of the mosquitoes in the window trap was not reduced by the use of an Olyset Plus net in the bedroom relative to an Olyset net., Conclusion: Mosquitoes entering bedrooms, even those susceptible to pyrethroids, were not killed by contact with an Olyset Plus LLIN. The enhanced numbers of An. gambiae or An. arabiensis collected in light-traps when a treated net is used requires further experimentation and may be because of a heightened escape reaction on the part of the mosquito.

Objective: To investigate knowledge and prevention practices regarding dengue and chikungunya amongst community members, as well as knowledge, treatment and diagnostic practices among healthcare workers., Method: We conducted a cross-sectional survey with 125 community members and 125 healthcare workers from 13 health facilities in six villages in the Hai district of Tanzania. A knowledge score was generated based on participant responses to a structured questionnaire, with a score of 40 or higher (of 80 and 50 total scores for community members and healthcare workers, respectively) indicating good knowledge. We conducted qualitative survey (n = 40) to further assess knowledge and practice regarding dengue and chikungunya fever., Results: 15.2% (n = 19) of community members had good knowledge regarding dengue, whereas 53.6%, (n = 67) of healthcare workers did. 20.3% (n = 16) of participants from lowland areas and 6.5% (n = 3) from highland areas had good knowledge of dengue (χ 2 = 4.25, P = 0.03). Only 2.4% (n = 3) of all participants had a good knowledge score for chikungunya. In the qualitative study, community members expressed uncertainty about dengue and chikungunya. Some healthcare workers thought that they were new diseases., Conclusion: There is insufficient knowledge regarding dengue and chikungunya fever among community members and healthcare workers. Health promotion activities on these diseases based on Ecological Health Mode components to increase knowledge and improve preventive practices should be developed., (© 2017 John Wiley & Sons Ltd.)

Background: A novel, insecticide-treated, durable wall lining (ITWL), which mimics indoor residual spraying (IRS), has been developed to provide prolonged vector control when fixed to the inner walls of houses. PermaNet ® ITWL is a polypropylene material containing non-pyrethroids (abamectin and fenpyroximate) which migrate gradually to the surface., Methods: An experimental hut trial was conducted in an area of pyrethroid-resistant Anopheles gambiae s.l. and Anopheles funestus s.s. to compare the efficacy of non-pyrethroid ITWL, long-lasting insecticidal nets (LLIN) (Interceptor ® ), pyrethroid ITWL (ZeroVector ® ), and non-pyrethroid ITWL + LLIN., Results: The non-pyrethroid ITWL produced relatively low levels of mortality, between 40-50% for An. funestus and An. gambiae, across all treatments. Against An. funestus, the non-pyrethroid ITWL when used without LLIN produced 47% mortality but this level of mortality was not significantly different to that of the LLIN alone (29%, P = 0.306) or ITWL + LLIN (35%, P = 0.385). Mortality levels for An. gambiae were similar to An. funestus with non-pyrethroid ITWL, producing 43% mortality compared with 26% for the LLIN. Exiting rates from ITWL huts were similar to the control and highest when the LLIN was present. An attempt to restrict mosquito access by covering the eave gap with ITWL (one eave open vs four open) had no effect on numbers entering. The LLIN provided personal protection when added to the ITWL with only 30% blood-fed compared with 69 and 56% (P = 0.001) for ITWL alone. Cone bioassays on ITWL with 30 min exposure after the trial produced mortality of >90% using field An. gambiae., Conclusions: Despite high mortality in bioassays, the hut trial produced only limited mortality which was attributed to pyrethroid resistance against the pyrethroid ITWL and low efficacy in the non-pyrethroid ITWL. Hut ceilings were left uncovered and may have served as a potential untreated refuge. By analogy to IRS campaigns, which also do not routinely treat ceilings, high community coverage with ITWL may still reduce malaria transmission. Restriction of eave gaps by 75% proved an inadequate barrier to mosquito entry. The findings represent the first 2 months after installation and do not necessarily predict long-term efficacy.

Introduction: Malaria prevalence has declined in the Kilimanjaro region of Tanzania over the past 10 years, particularly at lower altitudes. While this decline has been related to the scale-up of long-lasting insecticidal nets to achieve universal coverage targets, it has also been attributed to changes in environmental factors that are important for enabling and sustaining malaria transmission., Objectives: Herein, we apply spatial analytical approaches to investigate the impact of environmental and demographic changes, including changes in temperature, precipitation, land cover, and population density, on the range of the major malaria vector species Anopheles arabiensis in two districts of Tanzania, situated on the southern slope of Mount Kilimanjaro. These models are used to identify environmental changes that have occurred over a 10-year period and highlight the implications for malaria transmission in this highland region., Methods: Entomological data were collected from the Hai and Lower Moshi districts of Tanzania in 2001-2004 and 2014-2015. Vector occurrence data were applied alongside satellite remote sensing indices of climate and land cover, and gridded population data, to develop species distribution models for An. arabiensis for the 2004 and 2014 periods using maximum entropy. Models were compared to assess the relative contribution of different environmental and demographic factors to observed trends in vector species distribution in lowland and highland areas., Results: Changes in land cover were observed in addition to increased population densities, increased warm season temperature, and decreased wetness at low altitudes. The predicted area and extent of suitable habitat for An. arabiensis declined across the study area over the 10-year period, with notable contraction at lower altitudes, while species range in higher altitude zones expanded. Importantly, deforestation and warmer temperatures at higher altitudes may have created stable areas of suitable vector habitat in the highlands capable of sustaining malaria transmission., Conclusion: We show that environmental changes have had an important influence on the distribution of malaria vector species in a highland area of northern Tanzania. Highland areas may be at continued risk for sporadic malaria outbreaks despite the overall range contraction of principal vector species at lower altitudes, where malaria transmission remains at low intensity.

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 insecticide-treated 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.

Little is known about the presence and distribution of Aedes mosquitoes in northern Tanzania despite the occurence of viruses transmitted by these mosquitoes such as Chikungunya virus (CHIKV) and Dengue virus (DENV) in the region. Adult and larval mosquitoes were collected from rural and urban settings across a wide range of altitudes in the Kilimanjaro Region using the Mosquito Magnet CO2 Trap for collection of adults and old tires for breeding of larvae. Polymerase chain reaction assays were performed on captured adult mosquitoes to detect the presence of CHIKV and DENV. A total of 2609 Aedes aegypti adult mosquitoes were collected; no other Aedes species larvae were found. Mosquito yields were significantly higher in urban settings than rural settings (26.5 vs. 1.9 mosquitoes per day, p = 0.037). A total of 6570 Ae. aegypti larvae were collected from old tires; no other Aedes species larvae were found. Of the 2609 adult mosquitoes collected, none tested positive for CHIKV or DENV. As far as we are aware, this paper reports for the first time the presence of Ae. aegypti in the Kilimanjaro Region of northern Tanzania. Although CHIKV and DENV were not isolated from any of the collected mosquitoes in this study, the apparent absence of other Aedes species in the area suggests that Ae. aegypti is the primary local vector of these infections.

Background: In spite of increasing reports of dengue and chikungunya activity in Tanzania, limited research has been done to document the general epidemiology of dengue and chikungunya in the country. This study aimed at determining the sero-prevalence and prevalence of acute infections of dengue and chikungunya virus among participants presenting with malaria-like symptoms (fever, headache, rash, vomit, and joint pain) in three communities with distinct ecologies of north-eastern Tanzania., Methods: Cross sectional studies were conducted among 1100 participants (aged 2-70 years) presenting with malaria-like symptoms at health facilities at Bondo dispensary (Bondo, Tanga), Hai hospital (Hai, Kilimanjaro) and TPC hospital (Lower Moshi). Participants who were malaria negative using rapid diagnostic tests (mRDT) were screened for sero-positivity towards dengue and chikungunya Immunoglobulin G and M (IgG and IgM) using ELISA-based kits. Participants with specific symptoms defined as probable dengue and/or chikungunya by WHO (fever and various combinations of symptoms such as headache, rash, nausea/vomit, and joint pain) were further screened for acute dengue and chikungunya infections by PCR., Results: Out of a total of 1100 participants recruited, 91.2 % (n = 1003) were malaria negative by mRDT. Out of these, few of the participants (<5 %) were dengue IgM or IgG positive. A total of 381 participants had fever out of which 8.7 % (33/381) met the defined criteria for probable dengue, though none (0 %) was confirmed to be acute cases. Chikungunya IgM positives among febrile participants were 12.9 % (49/381) while IgG positives were at 3.7 % (14/381). A total of 74.2 % (283/381) participants met the defined criteria for probable chikungunya and 4.2 % (11/263) were confirmed by PCR to be acute chikungunya cases. Further analyses revealed that headache and joint pain were significantly associated with chikungunya IgM seropositivity., Conclusion: In north-eastern Tanzania, mainly chikungunya virus appears to be actively circulating in the population. Continuous surveillance is needed to determine the contribution of viral infections of fever cases. A possible establishment of arboviral vector preventive control measures and better diagnosis of pathogens to avoid over-treatment of other diseases should be considered.

Experimental huts with veranda traps have been used in Tanzania since 1963 for the study of residual insecticides for use with insecticide-treated nets and indoor residual spraying. Mosquitoes are allowed unrestricted entry through the eaves to facilitate the collection of an estimable proportion of mosquitoes that attempt to exit through the eave gaps, which are left open on two sides of the hut. This study was designed to validate the use of eave baffles to funnel entry and to prevent mosquito escape, and to determine biting times of Anopheles arabiensis (Patton) (Diptera: Culicidae). Anopheles arabiensis and Culex quinquefasciatus (Say) (Diptera: Culicidae) were released into the room at 20.30 hours and collected the following morning from veranda traps, window traps and the room. Centers for Disease Control light traps hung overnight next to volunteers were emptied every 2 h to determine peak biting times. A total of 55% of An. arabiensis were trapped before 22.30 hours and the highest peak in 'biting' was recorded during 18.30-20.30 hours. Of the released An. arabiensis that exited into veranda traps, 7% were captured in veranda traps entered through baffles and 93% were captured in traps entered through unmodified eaves. When veranda screens were left open to allow for escape outdoors, recapture rates were 68% for huts with eave baffles and 39% for huts with unmodified eaves. The comparison of open eaves with baffled eaves validated the assumption that in huts of the traditional non-baffled design, 50% of mosquitoes escape through open eaves. Eave baffles succeeded in reducing the potential for mosquito exit and produced more precise estimates of effect., (© 2015 The Royal Entomological Society.)

Indoor residual spraying (IRS) combined with insecticide treated nets (ITN) has been implemented together in several sub-Saharan countries with inconclusive evidence that the combined intervention provides added benefit. The impact on malaria transmission was evaluated in a cluster randomised trial comparing two rounds of IRS with bendiocarb plus universal coverage ITNs, with ITNs alone in northern Tanzania. From April 2011 to December 2012, eight houses in 20 clusters per study arm were sampled monthly for one night with CDC light trap collections. Anopheles gambiae s.l. were identified to species using real time PCR Taq Man and tested for the presence of Plasmodium falciparum circumsporozoite protein. ITN and IRS coverage was estimated from household surveys. IRS coverage was more than 85% in two rounds of spraying in January and April 2012. Household coverage with at least one ITN per house was 94.7% after the universal coverage net campaign in the baseline year and the proportion of household with all sleeping places covered by LLIN was 50.1% decreasing to 39.1% by the end of the intervention year. An.gambiae s.s. comprised 80% and An.arabiensis 18.3% of the anopheline collection in the baseline year. Mean An.gambiae s.l. density in the ITN+IRS arm was reduced by 84% (95%CI: 56%-94%, p = 0.001) relative to the ITN arm. In the stratum of clusters categorised as high anopheline density at baseline EIR was lower in the ITN+IRS arm compared to the ITN arm (0.5 versus 5.4 per house per month, Incidence Rate Ratio: 0.10, 95%CI: 0.01-0.66, p-value for interaction <0.001). This trial provides conclusive evidence that combining carbamate IRS and ITNs produces major reduction in Anopheles density and entomological inoculation rate compared to ITN alone in an area of moderate coverage of LLIN and high pyrethroid resistance in An.gambiae s.s.

Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed. Pyriproxyfen is a juvenile hormone mimic that reduces fecundity and fertility of adult Anopheles mosquitoes when used as a contact insecticide. A long-lasting insecticidal net incorporating pyriproxyfen is under development. As wild, host-seeking females may succeed in blood-feeding at different intervals after initial contact with mosquito nets the aim of this study was to determine the effect that age and gonotrophic status (nulliparous or parous) and the interval between initial pyriproxyfen exposure and blood-feeding has in terms of subsequent reduced fecundity and fertility. Anopheles gambiae s.s. were exposed to pyriproxyfen LLIN for three minutes in WHO cone bioassays. Four regimens were tested with different blood-feeding intervals A-1 hour (nulliparous), B-1 hour (parous), C-24h (nulliparous), or D-120h (nulliparous) after pyriproxyfen exposure. Mosquito oviposition rate, fecundity and fertility of eggs were recorded for several days. All four treatment regimens produced levels of mortality similar to unexposed females. The overall reduction in reproductive rate of 99.9% for regimen A relative to the untreated net was primarily due to oviposition inhibition in exposed females (97%). Pyriproxyfen was equally effective against older parous mosquitoes and when blood-feeding was 24h after exposure. Regimen D produced a reduction in reproductive rate of 60.1% but this was of lesser magnitude than other regimens and was the only regimen that failed to reduce fertility of laid eggs, indicating the effects of pyriproxyfen exposure on reproduction are to some extent reversible as mosquitoes age. In an area of moderate to high mosquito net coverage a host-seeking mosquito is likely to contact a treated mosquito net before: (a) penetrating a holed net and blood-feeding shortly after exposure or, (b) be frustrated by intact nets before succeeding in blood-feeding on an unprotected individual the following night. Mosquito nets are an appropriate delivery system for pyriproxyfen, based on the large reductions in reproductive rate when blood-feeding between 1h and 24h after exposure. Combining with a pyrethroid should be an effective approach if susceptible mosquitoes are killed and resistant mosquitoes sterilized., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: Universal coverage with long-lasting insecticidal mosquito nets (LLIN) or indoor residual spraying (IRS) of houses remain the primary strategies for the control of mosquito vectors of malaria. Pyrethroid resistant malaria vectors are widespread throughout sub-Saharan Africa and new insecticides with different modes of action are urgently needed if malaria vector control is to remain effective. Indoxacarb is an oxadiazine insecticide that is effective as an oral and contact insecticide against a broad spectrum of agricultural pests and, due to its unique site of action, no cross-resistance has been detected through mechanisms associated with resistance to insecticides currently used in public health., Methods: WHO tunnel tests of host seeking mosquitoes were carried out as a forerunner to experimental hut trials, to provide information on dosage-dependent mortality, repellency, and blood-feeding inhibition. A dosage range of indoxacarb treated netting (100-1000 mg/m(2)) was tested against a pyrethroid susceptible strain of Anopheles gambiae. In addition, efficacy of indoxacarb 500 mg/m(2) was compared with a standard pyrethroid formulation against pyrethroid susceptible and resistant Culex quinquefasciatus. Dosages between 25 and 300 mg/m(2) indoxacarb were tested in tunnel tests and in ball-frame bioassays as mixtures with alphacypermethrin 25 mg/m(2) and were compared with singly applied treatments against an insectary reared pyrethroid resistant strain of Cx. quinquefasciatus originally collected in Cotonou, Benin., Results: There was a dosage-dependent response in terms of indoxacarb induced mortality, with dosages >100 mg/m(2) producing the best mortality response. In tunnel tests indoxacarb 500 mg/m(2) exceeded WHOPES thresholds with >80 % mortality of adult An. gambiae and blood-feeding inhibition of 75 %. No cross-resistance to indoxacarb was detected through mechanisms associated with resistance to pyrethroid insecticides and was equally effective against susceptible and resistant strains of Cx. quinquefasciatus. Indoxacarb 500 mg/m(2) killed 75 % of pyrethroid resistant Cx. quinquefasciatus compared with only 21 % mortality with alphacypermethrin 40 mg/m(2). Mixtures of indoxacarb with pyrethroid produced an additive response for both mortality and blood-feeding inhibition. The best performing mixture (indoxacarb 200 mg/m(2) + alphacypermethrin 25 mg/m(2)) killed 83 % of pyrethroid resistant Cx. quinquefasciatus and reduced blood-feeding by 88 %, while alphacypermethrin only killed 36 % and inhibited blood-feeding by 50 %., Conclusions: New insecticides with different modes of action to those currently used in mosquito vector control are urgently needed. Indoxacarb shows great promise as a mixture with a pyrethroid and should be evaluated in experimental hut trials to determine performance against wild free-flying, pyrethroid resistant An. gambiae and wash-resistant formulations developed.

Background: Insecticide-treated nets are the primary method of preventing malaria. To remain effective, the pyrethroid insecticide must withstand multiple washes over the lifetime of the net. ICON(®) Maxx is a 'dip-it-yourself' kit for long-lasting treatment of polyester nets. The twin-sachet kit contains a slow-release capsule suspension of lambda-cyhalothrin plus binding agent. To determine whether ICON Maxx meets the standards required by the World Health Organization Pesticide Evaluation Scheme (WHOPES), the efficacy and wash fastness of ICON Maxx was evaluated against wild, free-flying anopheline mosquitoes., Methods: ICON Maxx was subjected to bioassay evaluation and experimental hut trial against pyrethroid-susceptible Anopheles gambiae, Anopheles arabiensis and Anopheles funestus. Mosquito mortality, blood feeding inhibition and personal protection were compared between untreated nets, conventional lambda-cyhalothrin treated nets (CTN) washed either four times (cut-off threshold) or 20 times, and ICON Maxx-treated nets either unwashed or washed 20 times., Results: In bioassay, ICON Maxx demonstrated superior wash resistance to the CTN. In the experimental hut trial, ICON Maxx killed 75 % of An. funestus, 71 % of An. gambiae and 47 % of An. arabiensis when unwashed and 58, 66 and 42 %, respectively, when 20 times washed. The CTN killed 52 % of An. funestus, 33 % of An. gambiae and 30 % of An. arabiensis when washed to the cut-off threshold of four washes and 40, 40 and 36 %, respectively, when 20 times washed. Percentage mortality with ICON Maxx 20 times washed was similar (An. funestus) or significantly higher (An. gambiae, An. arabiensis) than with CTN washed to the WHOPES cut-off threshold. Blood-feeding inhibition with ICON Maxx 20 times washed was similar to the CTN washed to cut-off for all three species. Personal protection was significantly higher with ICON Maxx 20 times washed (66-79 %) than with CTN washed to cut-off (48-60 %)., Conclusions: Nets treated with ICON Maxx and washed 20 times met the approval criteria set by WHOPES for Phase II trials in terms of mortality and blood-feeding inhibition. This finding raises the prospect of conventional polyester nets and other materials being made long-lastingly insecticidal through simple dipping in community or home, and thus represents a major advance over conventional pyrethroid treatments.

Background: Insecticide treated nets (ITNs) and indoor residual spraying (IRS) are effective vector control tools that protect against malaria. There is conflicting evidence regarding whether using ITNs and IRS in combination provides additional benefit over using either of these methods alone. This study investigated factors that may modify the effect of the combined use of IRS and ITNs compared to using ITNs alone on malaria infection prevalence., Methods: Secondary analysis was carried out on data from a cluster randomised trial in north-west Tanzania. 50 clusters received ITNs from a universal coverage campaign; of these 25 were randomly allocated to additionally receive two rounds of IRS in 2012. In cross-sectional household surveys children 0.5-14 years old were tested for Plasmodium falciparum infections (PfPR) two, six and ten months after the first IRS round., Results: IRS protected those sleeping under nets (OR = 0.38, 95%CI 0.26-0.57) and those who did not (OR = 0.43, 95%CI 0.29-0.63). The protective effect of IRS was not modified by community level ITN use (ITN use<50%, OR = 0.39, 95%CI 0.26-0.59; ITN use> = 50%, OR = 0.46, 95%CI 0.28-0.74). The additional protection from IRS was similar in low (<10% PfPR, OR = 0.38, 95%CI 0.19-0.75) and high transmission areas (≥10% PfPR, OR = 0.34, 95%CI 0.18-0.67). ITN use was protective at the individual-level regardless of whether the village had been sprayed (OR = 0.83, 95%CI 0.70-0.98). Living in a sprayed village was protective regardless of whether the individual slept under an ITN last night (OR = 0.41, 95%CI 0.29-0.58)., Interpretation: Implementing IRS in addition to ITNs was beneficial for individuals from villages with a wide range of transmission intensities and net utilisation levels. Net users received additional protection from IRS. ITNs were providing some individual protection, even in this area with high levels of pyrethroid insecticide resistance. These results demonstrate that there is a supplementary benefit of IRS even when ITNs are effective., Trial Registration: ClinicalTrials.gov NCT01697852.

Background: The rapid selection of pyrethroid resistance throughout sub-Saharan Africa is a serious threat to malaria vector control. Chlorfenapyr is a pyrrole insecticide which shows no cross resistance to insecticide classes normally used for vector control and is effective on mosquito nets under experimental hut conditions. Unlike neurotoxic insecticides, chlorfenapyr owes its toxicity to disruption of metabolic pathways in mitochondria that enable cellular respiration. A series of experiments explored whether standard World Health Organization (WHO) guidelines for evaluation of long-lasting insecticidal nets, developed through testing of pyrethroid insecticides, are suitable for evaluation of non-neurotoxic insecticides., Methods: The efficacy of WHO recommended cone, cylinder and tunnel tests was compared for pyrethroids and chlorfenapyr. To establish bioassay exposure times predictive of insecticide-treated net (ITN) efficacy in experimental hut trials, standard three-minute bioassays of pyrethroid and chlorfenapyr ITNs were compared with longer exposures. Mosquito behaviour and response to chlorfenapyr ITN in bioassays conducted at night were compared to day and across a range of temperatures representative of highland and lowland transmission., Results: Standard three-minute bioassay of chlorfenapyr produced extremely low levels of mortality compared to pyrethroids. Thirty-minute day-time bioassay produced mortality closer to hut efficacy of chlorfenapyr ITN but still fell short of the WHO threshold. Overnight tunnel test with chlorfenapyr produced 100% mortality and exceeded the WHO threshold of 80%. The endogenous circadian activity rhythm of anophelines results in inactivity by day and raised metabolism and flight activity by night. A model which explains improved toxicity of chlorfenapyr ITN when tested at night, and during the day at higher ambient temperature, is that activation of chlorfenapyr and disruption of respiratory pathways is enhanced when the insect is more metabolically and behaviourally active., Conclusions: Testing according to current WHO guidelines is not suitable for certain types of non-neurotoxic insecticide which, although highly effective in field trials, would be overlooked at the screening stage of evaluation through bioassay. Testing methods must be tailored to the characteristics and mode of action of each insecticide class. The WHO tunnel test on night-active anophelines is the most reliable bioassay for identifying the toxicity of novel insecticides.