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Mosquito-transmitted diseases are a major threat to health in sub-Saharan Africa, but could be reduced through modifications to the built environment. Here we report findings from a major workshop held to identify the research gaps in this area, namely: (1) evidence of the health benefits to changes to the built environment, (2) understanding how mosquitoes enter buildings, (3) novel methods for reducing mosquito-house entry, (4) sustainable approaches for reducing mosquito habitats, (5) case studies of micro-financing for healthy homes and (6) methods for increasing scale-up. Multidisciplinary research is essential to build out mosquito-transmitted diseases, and not build them in.

Five years of sustained indoor residual spraying (IRS) of insecticide from 2014 to 2019, first using a carbamate followed by an organophosphate, was associated with a marked reduction in the incidence of malaria in five districts of Uganda. We assessed changes in malaria incidence over an additional 21 months, corresponding to a change in IRS formulations using clothianidin with and without deltamethrin. Using enhanced health facility surveillance data, our objectives were to 1) estimate the impact of IRS on monthly malaria case counts at five surveillance sites over a 6.75 year period, and 2) compare monthly case counts at five facilities receiving IRS to ten facilities in neighboring districts not receiving IRS. For both objectives, we specified mixed effects negative binomial regression models with random intercepts for surveillance site adjusting for rainfall, season, care-seeking, and malaria diagnostic. Following the implementation of IRS, cases were 84% lower in years 4–5 (adjusted incidence rate ratio [aIRR] = 0.16, 95% CI 0.12–0.22), 43% lower in year 6 (aIRR = 0.57, 95% CI 0.44–0.74), and 39% higher in the first 9 months of year 7 (aIRR = 1.39, 95% CI 0.97–1.97) compared to pre-IRS levels. Cases were 67% lower in IRS sites than non-IRS sites in year 6 (aIRR = 0.33, 95% CI 0.17–0.63) but 38% higher in the first 9 months of year 7 (aIRR = 1.38, 95% CI 0.90–2.11). We observed a resurgence in malaria to pre-IRS levels despite sustained IRS. The timing of this resurgence corresponded to a change of active ingredient. Further research is needed to determine causality.

Background\ud The World Health Organization (WHO) recommends artemisinin‐based combination therapies (ACTs) to treat uncomplicated Plasmodium falciparum malaria. Concerns about artemisinin resistance have led to global initiatives to develop new partner drugs to protect artemisinin derivatives in ACT. Pyronaridine‐artesunate is a novel ACT.\ud \ud Objectives\ud To evaluate the efficacy of pyronaridine‐artesunate compared to alternative ACTs for treating people with uncomplicated P falciparum malaria, and to evaluate the safety of pyronaridine‐artesunate and other pyronaridine treatments compared to alternative treatments.\ud \ud Search methods\ud We searched the Cochrane Infectious Diseases Group Specialized Register; Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE; Embase; and LILACS. We also searched ClinicalTrials.gov, the WHO International Clinical Trials Registry Platform, and the ISRCTN registry for ongoing or recently completed trials. The date of the last search was 27 October 2021.\ud \ud Selection criteria\ud For the efficacy analysis, we included randomized controlled trials (RCTs) of pyronaridine‐artesunate for treating uncomplicated P falciparum malaria. For the safety analysis, we included RCTs that used pyronaridine alone or in combination with any other antimalarials. In addition to these analyses, we conducted a separate systematic review summarizing data on safety from non‐randomized studies (NRS) of any patient receiving pyronaridine (NRS safety review). \ud \ud Data collection and analysis\ud Two review authors independently extracted all data and assessed the certainty of the evidence. We meta‐analysed data to calculate risk ratios (RRs) for treatment failures between comparisons, and for safety outcomes between and across comparisons.\ud \ud Main results\ud We included 10 relevant RCTs. Seven RCTs were co‐funded by Shin Poong Pharmaceuticals, and three were funded by government agencies.\ud \ud Efficacy analysis (RCTs)\ud \ud For the efficacy analysis, we identified five RCTs comprising 5711 participants. This included 4465 participants from 13 sites in Africa, and 1246 participants from five sites in Asia. The analysis included 541 children aged less than five years. Overall, pyronaridine‐artesunate had a polymerase chain reaction (PCR)‐adjusted treatment failure rate of less than 5%. We evaluated pyronaridine‐artesunate versus the following.\ud \ud • Artemether‐lumefantrine. Pyronaridine artesunate may perform better for PCR‐adjusted failures at day 28 (RR 0.59, 95% confidence interval (CI) 0.26 to 1.31; 4 RCTs, 3068 participants, low‐certainty evidence); for unadjusted failures at day 28 (RR 0.27, 95% CI 0.13 to 0.58; 4 RCTs, 3149 participants, low‐certainty evidence); and for unadjusted failures at day 42 (RR 0.61, 95% CI 0.46 to 0.82; 4 RCTs, 3080 participants, low‐certainty evidence). For PCR‐adjusted failures at day 42, there may be little or no difference between groups (RR 0.86, 95% CI 0.49 to 1.51; 4 RCTs, 2575 participants, low‐certainty evidence).\ud \ud • Artesunate‐amodiaquine. Pyronaridine artesunate may perform better for PCR‐adjusted failures at day 28 (RR 0.55, 95% CI 0.11 to 2.77; 1 RCT, 1245 participants, low‐certainty evidence); probably performs better for unadjusted failures at day 28 (RR 0.49, 95% CI 0.30 to 0.81; 1 RCT, 1257 participants, moderate‐certainty evidence); may make little or no difference for PCR‐adjusted failures at day 42 (RR 0.98, 95% CI 0.20 to 4.83; 1 RCT, 1091 participants, low‐certainty evidence); and probably makes little or no difference for unadjusted failures at day 42 (RR 0.98, 95% CI 0.78 to 1.23; 1 RCT, 1235 participants, moderate‐certainty evidence).\ud \ud • Mefloquine plus artesunate. Pyronaridine artesunate may perform better for PCR‐adjusted failures at day 28 (RR 0.37, 95% CI 0.13 to 1.05; 1 RCT, 1117 participants, low‐certainty evidence); probably performs better for unadjusted failures at day 28 (RR 0.36, 95% CI 0.17 to 0.78; 1 RCT, 1120 participants, moderate‐certainty evidence); may make little or no difference for unadjusted failures at day 42 (RR 0.84, 95% CI 0.54 to 1.31; 1 RCT, 1059 participants, low‐certainty evidence); but may lead to higher PCR‐adjusted failures at day 42 (RR 1.80, 95% CI 0.90 to 3.57; 1 RCT, 1037 participants, low‐certainty evidence).\ud \ud Safety analysis (RCTs)\ud \ud For the RCT safety analysis, we identified eight RCTs, one of which was delineated by study site, comparing pyronaridine‐artesunate to other antimalarials. Pyronaridine‐artesunate was associated with raised liver enzymes compared to other antimalarials: alanine aminotransferase (ALT) (RR 3.59, 95% CI 1.76 to 7.33; 8 RCTS, 6669 participants, high‐certainty evidence) and aspartate transaminase (AST) (RR 2.22, 95% CI 1.12 to 4.41; 8 RCTs, 6669 participants, moderate‐certainty evidence). No such effect was demonstrated with bilirubin (RR 1.03, 95% CI 0.49 to 2.18; 7 RCTs, 6384 participants, moderate‐certainty evidence). There was one reported case in which raised ALT occurred with raised bilirubin. No study reported severe drug‐induced liver injury. Electrocardiograph (ECG) abnormalities were less common with pyronaridine‐artesunate compared to other antimalarials. We identified no other safety concerns.\ud \ud NRS safety review\ud \ud A review on safety in NRS allowed us to increase the population within which safety was assessed. We included seven studies with 9546 participants: five single‐arm observational studies, one cohort event monitoring study, and one dose‐escalation study. All studies provided data on adverse event frequency, with a small number of participants experiencing serious adverse events and adverse effects related to pyronaridine: serious adverse events average 0.37%; drug‐related 9.0%. In two studies reporting elevations in liver enzymes, small percentages of participants (2.4% and 14.1% respectively) experienced increases in either ALT, AST, or bilirubin on day 7; however, these were small increases that returned to normal by day 42. \ud \ud Authors' conclusions\ud Pyronaridine‐artesunate was efficacious against uncomplicated P falciparum malaria; achieved a PCR‐adjusted treatment failure rate of less than 5% at days 28 and 42; and may be at least as good as, or better than, other marketed ACTs.\ud \ud Pyronaridine‐artesunate increases the risk of episodes of abnormally raised ALT. The observational data did not signal an excess of clinically important adverse effects.

Background Agricultural pesticides may exert strong selection pressures on malaria vectors during the aquatic life stages and may contribute to resistance in adult mosquitoes. This could reduce the performance of key vector control interventions such as indoor-residual spraying and insecticide-treated nets. The aim of this study was to investigate effects of agrochemicals on susceptibility and fitness of the malaria vectors across farming areas in Tanzania. Methods An exploratory mixed-methods study was conducted to assess pesticide use in four villages (V1–V4) in south-eastern Tanzania. Anopheles gambiae (s.l.) larvae were collected from agricultural fields in the same villages and their emergent adults examined for insecticide susceptibility, egg-laying and wing lengths (as proxy for body size). These tests were repeated using two groups of laboratory-reared An. arabiensis, one of which was pre-exposed for 48 h to sub-lethal aquatic doses of agricultural pesticides found in the villages. Results Farmers lacked awareness about the linkages between the public health and agriculture sectors but were interested in being more informed. Agrochemical usage was reported as extensive in V1, V2 and V3 but minimal in V4. Similarly, mosquitoes from V1 to V3 but not V4 were resistant to pyrethroids and either pirimiphos-methyl or bendiocarb, or both. Adding the synergist piperonyl butoxide restored potency of the pyrethroids. Pre-exposure of laboratory-reared mosquitoes to pesticides during aquatic stages did not affect insecticide susceptibility in emergent adults of the same filial generation. There was also no effect on fecundity, except after pre-exposure to organophosphates, which were associated with fewer eggs and smaller mosquitoes. Wild mosquitoes were smaller than laboratory-reared ones, but fecundity was similar. Conclusions Safeguarding the potential of insecticide-based interventions requires improved understanding of how agricultural pesticides influence important life cycle processes and transmission potential of mosquito vectors. In this study, susceptibility of mosquitoes to public health insecticides was lower in villages reporting frequent use of pesticides compared to villages with little or no pesticide use. Variations in the fitness parameters, fecundity and wing length marginally reflected the differences in exposure to agrochemicals and should be investigated further. Pesticide use may exert additional life cycle constraints on mosquito vectors, but this likely occurs after multi-generational exposures. Graphical Abstract

Background To sustain high universal Long-Lasting Insecticidal Nets (LLINs) coverage, affordable nets that provide equivalent or better protection than standard LLINs, are required. Test facilities evaluating new LLINs require compliance to Good Laboratory Practice (GLP) standards to ensure the quality and integrity of test data. Following GLP principles allows for the reconstruction of activities during the conduct of a study and minimizes duplication of efficacy testing. This case study evaluated the efficacy of two LLINs: SafeNet NF® and SafeNet® LLIN. Methods The study was conducted according to GLP principles and followed World Health Organization guidelines for evaluating LLINs. The LLINs were assessed in experimental huts against wild, pyrethroid-resistant Anopheles arabiensis mosquitoes. Nets were either unwashed or washed 20 times and artificially holed to simulate a used torn net. Blood-feeding inhibition and mortality were compared with a positive control (Interceptor® LLIN) and an untreated net. Results Mosquito entry in the huts was reduced compared to negative control for the unwashed SafeNet NF, washed Safenet LLIN and the positive control arms. Similar exiting rates were found for all the treatment arms. Significant blood-feeding inhibition was only found for the positive control, both when washed and unwashed. All insecticide treatments induced significantly higher mortality compared to an untreated net. Compared to the positive control, the washed and unwashed SafeNet NF® resulted in similar mortality. For the SafeNet® LLINs the unwashed net had an equivalent performance, but the mortality for the washed net was significantly lower than the positive control. Internal audits of the study confirmed that all critical phases complied with Standard Operating Procedures (SOPs) and the study plan. The external audit confirmed that the study complied with GLP standards. Conclusions SafeNet NF® and SafeNet® LLIN offered equivalent protection to the positive control (Interceptor® LLIN). However, further research is needed to investigate the durability, acceptability, and residual efficacy of these nets in the community. This study demonstrated that GLP-compliant evaluation of LLINs can be successfully conducted by African research institutions.

Long-lasting insecticide-treated nets (LLINs) play a crucial role in preventing malaria transmission. LLINs should remain effective for at least three years, even after repeated washings. Currently, monitoring insecticides in LLINs is cumbersome, costly, and requires specialized equipment and hazardous solvents. Our aim was to develop a simple, high-throughput and low-resource method for measuring insecticides in LLINs. To extract insecticides, polyethylene-LLIN samples were heated at 85 °C for 45 min in a non-hazardous solvent mix containing dicyclohexylphthalate as an internal standard. The extraction solvent was reduced from 50 to 5 ml using a 0.2 g sample, 90% smaller than the recommended sample size. By optimizing HPLC chromatography, we simultaneously detected pyrethroid and pyriproxyfen insecticides with high sensitivity in LLIN's extract. The method can quantify levels ≥ 0.0015% permethrin, 0.00045% alpha-cypermethrin and 0.00025% pyriproxyfen (w/w) in polyethylene, allowing for insecticide tracking before and after the use of LLINs. This method can be used to assess LLINs with 1% pyriproxyfen (pyriproxyfen-LLIN) or 2% permethrin (Olyset® Net), 1% pyriproxyfen and 2% permethrin (Olyset® Duo), or 0.55% pyriproxyfen and 0.55% alpha-cypermethrin (Royal Gaurd®). One can run 120 samples (40 nets) simultaneously with high precision and accuracy, improving throughput and reducing labour, costs, and environmental impact.

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.

Vector control programs worldwide are facing the challenge of mosquitoes becoming resistant to available insecticides. Larviciding is a crucial preventative measure for dengue control but data on insecticide resistance of larval Ae. aegypti in the Middle Eastern Region are limited. This study assesses the susceptibility status of Ae. aegypti collected from the two most important dengue foci in Saudi Arabia, Jeddah and Makkah, to important chemical and biological larvicides; the organophosphate temephos and Bacillus thuringiensis israelensis, Bti). Whilst worldwide, and particularly in Latin America, high-level resistance to temephos is common, Jeddah and Makkah populations exhibited full susceptibility to both temephos and Bti. These data suggest each can be considered by vector control programs for preventative dengue control in the region, as part of temporal rotations or spatial mosaics to manage insecticide resistance.\ud Key words: Mosquito larvae, Larval bioassay, Bti, temephos

Culex mosquitoes particularly Culex quinquefasciatus are important arboviral and filariasis vectors, however despite this important epidemiological role, there is still a paucity of data on their bionomics. The present study was undertaken to assess the insecticide resistance status of Cx. quinquefasciatus populations from four districts of Yaoundé (Cameroon). All Culex quinquefasciatus populations except one displayed high resistance to bendiocarb and malathion with mortalities ranging from 0 to 89% while high resistance intensity against both permethrin and deltamethrin was recorded. Molecular analyses revealed high frequencies of the ACE-1 G119S mutation (ranging from 0 to 33%) and kdr L1014F allele (ranging from 55 to 74%) in all Cx. quinquefasciatus populations. Significant overexpression was detected for cytochrome P450s genes CYP6AA7 and CYP6Z10, as well as for Esterase A and Esterase B genes. The total cuticular hydrocarbon content, a proxy of cuticular resistance, was significantly increased (compared to the S-lab strain) in one population. The study confirms strong insecticide resistance mediated by different mechanisms in Cx. quinquefasciatus populations from the city of Yaoundé. The expansion of insecticide resistance in Culex populations could affect the effectiveness of current vector control measures and stress the need for the implementation of integrated vector control strategies in urban settings.

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

Long-lasting insecticidal nets (LLINs) are the foundation of malaria control but resistance of mosquito vectors to pyrethroids threatens their effectiveness. We embedded a cluster-randomised trial into Uganda's 2017-18 campaign to distribute LLINs. LLINs with piperonyl butoxide (PBO) reduced parasite prevalence more effectively than conventional LLINs (without PBO) for 18 months. Here, we report the final 25-month survey results.LLINEUP was a cluster-randomised trial conducted in 48 districts in eastern and western Uganda. 104 health subdistricts (clusters) without ongoing or planned indoor residual spraying with pirimiphos-methyl (Actellic, Basel, Switzerland) were eligible for inclusion in the trial. Clusters were randomly assigned to PBO LLINs (PermaNet 3.0 or Olyset Plus) and conventional LLINs (PermaNet 2.0 or Olyset Net) with proportionate randomisation using STATA version 14.2. LLINs were delivered from March 25, 2017, to March 18, 2018. Between April 23, 2019, and Sept 13, 2019, community surveys were conducted in 50 randomly selected households per cluster; ten households per cluster were randomly selected for entomology surveys. Mosquitoes were collected in the morning from indoor surfaces of households using Prokopack aspirators. Due to COVID-19 restrictions, only 90 of the 104 clusters were surveyed at 25 months. The primary outcome was parasite prevalence by microscopy in children aged 2-10 years, assessed in the as-treated population, determined using the results from the 6-month household survey on the type of LLINs received in each cluster. This trial is registered with ISRCTN, ISRCTN17516395, and is now completed.In the as-treated analysis, two clusters were excluded (no predominant LLIN received) and four were reassigned; 40 PBO LLIN clusters (30 PermaNet 3.0, ten Olyset Plus) and 48 non-PBO LLIN (36 PermaNet 2.0, 12 Olyset Net) were included. Parasite prevalence was 17·1% (506 of 2958 participants) in the PBO group and 19·8% (701 of 3534) in the non-PBO group (prevalence ratio adjusted for baseline 0·80 [95% CI 0·69-0·93], p=0·0048). Comparing within-treatment group parasite prevalence to baseline, parasite prevalence ratios were lower in the PBO groups at all timepoints, but the difference was greatest at 6 months (PBO LLINs parasite prevalence at baseline 28·8% [1001 of 3472, 95% CI 27·3-30·4] vs at 6 months 12·0% [361 of 3009, 10·9-13·2], prevalence ratio [PR] 0·43 [95% CI 0·36-0·52], p0·0001; non-PBO LLINs parasite prevalence at baseline 25·4% [1015 of 4004, 24·0-26·7] vs 6 months 14·8% [526 of 3551, 13·7-16·0], PR 0·60 [0·54-0·68], p0·0001) and 25 months (PBO LLINs parasite prevalence at 25 months 17·1% [506 of 2958, 15·8-18·5], PR 0·63 [95% CI 0·57-0·71], p0·0001; non-PBO LLINs parasite prevalence at 25 months 19·8% [701 of 3534, 18·5-21·2], PR 0·79 [0·73-0·86], p0·0001).In Uganda, PBO LLINs outperformed pyrethroid-only LLINs for 25 months. WHO concluded that PBO LLINs are more effective against malaria than non-PBO LLINs when resistance to pyrethroids is high and issued a conditional recommendation suggesting PBO LLINs should be deployed in areas of pyrethroid resistance.The Against Malaria Foundation, UK Department for International Development, Innovative Vector Control Consortium, and Bill and Melinda Gates Foundation.

Background There is a pressing need to improve understanding of how insecticide resistance affects the functional performance of insecticide-treated nets (ITNs). Standard WHO insecticide resistance monitoring assays are designed for resistance surveillance and do not necessarily provide insight into how different frequencies, mechanisms or intensities of resistance affect the ability of ITNs to reduce malaria transmission. Methods The current study presents some novel laboratory-based assays that attempt to better simulate realistic exposure of mosquitoes to ITNs and to quantify impact of exposure not only on instantaneous mortality, but also on blood-feeding and longevity, two traits that are central to transmission. The assays evaluated the performance of a standard ITN (Permanet® 2.0; Vestergaard Frandsen), a ‘next generation’ combination ITN with a resistance-breaking synergist (Permanet® 3.0) and an untreated net (UTN), against field-derived Anopheles gambiae sensu lato mosquitoes from Côte d’Ivoire exhibiting a 1500-fold increase in pyrethroid resistance relative to a standard susceptible strain. Results The study revealed that the standard ITN induced negligible instantaneous mortality against the resistant mosquitoes, whereas the resistance-breaking net caused high mortality and a reduction in blood-feeding. However, both ITNs still impacted long-term survival relative to the UTN. The impact on longevity depended on feeding status, with blood-fed mosquitoes living longer than unfed mosquitoes following ITN exposure. Exposure to both ITNs also reduced the blood-feeding success, the time spent on the net and blood-feeding duration, relative to the untreated net. Conclusion Although a standard ITN did not have as substantial instantaneous impact as the resistance-breaking net, it still had significant impacts on traits important for transmission. These results highlight the benefit of improved bioefficacy assays that allow for realistic exposure and consider sub- or pre-lethal effects to help assess the functional significance of insecticide resistance. Graphical Abstract

Understanding mosquito interaction with long-lastig insecticidal bednets is crucial in the development of more effective intervention methods to protect humans from malaria transmission. As such, a 240 × 240 × 1000 mm laboratory setup for the in-line recording of digital holograms and subsequent in-focus reconstruction and 3D localisation of mosquitoes is presented. Simple bednet background removal methods are used to accurately localise a mosquito obscured by a bednet in 3D coordinates. Simulations and physical data demonstrate that this\ud method is suitable for mosquitoes positioned 3−1000 mm behind a bednet. A novel post-processing technique, involving a cascade-correlation of a Tamura of Intensity focus metric extracted from digitally reconstructed scenes, accurately localises mosquitoes positioned 35−100 mm behind a bednet from a single digital hologram. The result\ud of this study is a scalable digital holographic methodology to examine mosquito-bednet interaction in 3D at a level of accuracy previously only seen in 2D imaging of mosquitoes in a much smaller volume.

Background There is an urgent need for insecticides with novel modes of action against mosquito vectors. Broflanilide is a meta-diamide, discovered and named Tenebenal™ by Mitsui Chemicals Agro, Inc., which has been identified as a candidate insecticide for use in public health products. Methods To evaluate its potential for use in public health, Tenebenal™ was screened using an array of methodologies against Anopheles and Aedes strains. Initially it was assessed for intrinsic efficacy by topical application. Tarsal contact bioassays were then conducted to further investigate its efficacy, as well as its potency and speed of action. The potential of the compound for use in indoor residual spray (IRS) applications was investigated by testing the residual efficacy of a prototype IRS formulation on a range of typical house building substrates, and its potential for use in long-lasting insecticidal nets (LLIN) was tested using dipped net samples. Finally, bioassays using well-characterized insecticide-resistant mosquito strains and an in silico screen for mutations in the insecticide’s target site were performed to assess the risk of cross-resistance to Tenebenal™. Results Tenebenal™ was effective as a tarsal contact insecticide against both Aedes and Anopheles mosquitoes, with no apparent cross-resistance caused by mechanisms that have evolved to insecticides currently used in vector control. Topical application showed potent intrinsic activity against a Kisumu reference strain and an insecticide-resistant strain of Anopheles gambiae. Applied to filter paper in a WHO tube bioassay, Tenebenal™ was effective in killing 100% of susceptible and resistant strains of An. gambiae and Aedes aegypti at a concentration of 0.01%. The discriminating concentration of 11.91 µg/bottle shows it to be very potent relative to chemistries previously identified as having potential for vector control. Mortality occurs within 24 h of exposure, 80% of this mortality occurring within the first 10 h, a speed of kill somewhat slower than seen with pyrethroids due to the mode of action. The potential of Tenebenal™ for development in LLIN and IRS products was demonstrated. At least 12 months residual efficacy of a prototype IRS formulation applied at concentrations up to 200 mg of AI/sq m was demonstrated on a range of representative wall substrates, and up to 18 months on more inert substrates. A dipped net with an application rate of around 2 g/sq m Tenebenal™ killed 100% of exposed mosquitoes within a 3-min exposure in a WHO cone test. Conclusions Tenebenal™ is a potent insecticide against adult Aedes and Anopheles mosquitoes, including strains resistant to classes of insecticide currently used in vector control. The compound has shown great potential in laboratory assessment and warrants further investigation into development for the control of pyrethroid-resistant mosquitoes.

Background While Iran is on the path to eliminating malaria, the disease with 4.9 million estimated cases and 9300 estimated deaths in 2018 remains a serious health problem in the World Health Organization (WHO) Eastern Mediterranean Region. Anopheles stephensi is the main malaria vector in Iran and its range extends from Iraq to western China. Recently, the vector invaded new territories in Sri Lanka and countries in the Horn of Africa. Insecticide resistance in An. stephensi is a potential issue in controlling the spread of this vector. Methods Data were collated from national and international databases, including PubMed, Google Scholar, Scopus, ScienceDirect, SID, and IranMedex using appropriate search terms. Results Indoor residual spaying (IRS) with DDT was piloted in Iran in 1945 and subsequently used in the malaria eradication programme. Resistance to DDT in An. stephensi was detected in Iran, Iraq, Pakistan, and Saudi Arabia in the late 1960s. Malathion was used for malaria control in Iran in 1967, then propoxur in 1978, followed by pirimiphos-methyl from 1992 to 1994. The pyrethroid insecticide lambda-cyhalothrin was used from 1994 to 2003 followed by deltamethrin IRS and long-lasting insecticidal nets (LLINs). Some of these insecticides with the same sequence were used in other malaria-endemic countries of the region. Pyrethroid resistance was detected in An. stephensi in Afghanistan in 2010, in 2011 in India and in 2012 in Iran. The newly invaded population of An. stephensi in Ethiopia was resistant to insecticides of all four major insecticide classes. Different mechanisms of insecticide resistance, including metabolic and insecticide target site insensitivity, have been developed in An. stephensi. Resistance to DDT was initially glutathione S-transferase based. Target site knockdown resistance was later selected by pyrethroids. Esterases and altered acetylcholinesterase are the underlying cause of organophosphate resistance and cytochrome p450s were involved in pyrethroid metabolic resistance. Conclusions Anopheles stephensi is a major malaria vector in Iran and many countries in the region and beyond. The species is leading in terms of development of insecticide resistance as well as developing a variety of resistance mechanisms. Knowledge of the evolution of insecticide resistance and their underlying mechanisms, in particular, are important to Iran, considering the final steps the country is taking towards malaria elimination, but also to other countries in the region for their battle against malaria. This systematic review may also be of value to countries and territories newly invaded by this species, especially in the Horn of Africa, where the malaria situation is already dire.

Currently, dengue control relies largely on reactive vector control programmes. Proactive vector-control using a rational, well-balanced integrated vector management approach may prove more successful for dengue control. As part of the development of a cluster randomized controlled epidemiological trial, a study was conducted in Johor Bahru, Malaysia. The study included one control site (three buildings) and three intervention sites which were treated as follows: targeted outdoor residual spraying only (TORS site, two buildings); deployment of autodissemination devices only (ADD site, four buildings); and the previous two treatments combined (TORS + ADD site, three buildings). The primary entomological measurement was per cent of positive ovitraps—ovitrap index (OI). The effect of each intervention on OI was analyzed by a modified ordinary least squares regression model. Relative to the control site, the TORS and ADD sites showed a reduction in the Aedes OI (−6.5%, P = 0.04 and −8.3%, P = 0.10, respectively). Analysis by species showed that, relative to control, the Ae. aegypti OI was lower in ADD (−8.9%, P = 0.03) and in TORS (−10.4%, P = 0.02). No such effect was evident in the TORS + ADD site. The present study provides insights into the methods to be used for the main trial. The combination of multiple insecticides with different modes of action in one package is innovative, although we could not demonstrate the additive effect of TORS + ADD. Further work is required to strengthen our understanding of how these interventions impact dengue vector populations and dengue transmission.

The use of agrochemicals in vegetable production could influence the selection for insecticide resistance in malaria vectors. Unfortunately, there is a dearth of information on the potential contribution of agrochemicals to insecticide resistance in Anopheles mosquitoes breeding on vegetable farms in southern Benin. A Knowledge, Attitudes and Practices study was conducted with 75 vegetable farmers from Houeyiho and Seme to determine the main agrochemicals used in vegetable production, and the concentration and frequency of application, among other details. Mosquitoes and breeding water were sampled from the farms for analysis. Bioassays were conducted on mosquitoes, while breeding water was screened for heavy metal and pesticide residue contamination. Lambda-cyhalothrin was the main insecticide (97.5%) used by farmers, and Anopheles coluzzii was the main mosquito identified. This mosquito species was resistant (30–63% mortality rate) to λ-cyhalothrin. It was also observed that 16.7% of the examined breeding sites were contaminated with λ-cyhalothrin residues. Furthermore, copper contamination detected in mosquito breeding sites showed a positive correlation (r = 0.81; P = 0.0017) with mosquito resistance to λ-cyhalothrin. The presence of copper in λ-cyhalothrin-free breeding sites, where mosquitoes have developed resistance to λ-cyhalothrin, suggests the involvement of copper in the insecticide resistance of malaria vectors; this, however, needs further investigation.

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

Background The WHO cone test is one of three tests currently used to evaluate the efficacy of insecticide-treated bed nets (ITNs). It generates two test outputs, knockdown and 24-h mortality, both indicative of immediate toxicity but that reveal little about the nature of mosquito and ITN interaction or how results translate to real-world settings. Methods A human arm held 5 mm behind the net surface acted as a host attractant during cone tests and a smartphone was used to capture mosquito behaviour in the cone. Post-exposure blood feeding and survival for nine days were recorded; ingested blood meal size was determined by measuring excreted haematin. Four strains of Anopheles gambiae (insecticide susceptible: Kisumu and N’gousso; insecticide resistant: Banfora and VK7) were tested with and without the host attractant using untreated, Permanet 2.0 and Olyset nets. Video recordings were scan sampled every five seconds to record mosquito positions on either the net, in flight or in contact with the cone. Generalized estimating equations were used to analyse all data except survival within nine days which was analysed using Weighted Cox Regression. Results Net contact was the most frequently recorded behaviour in all Anopheles spp. strains on all nets. Adding the human host as attractant triggered excitatory behaviours: in all strains, the magnitude of net contact was significantly decreased compared to tests without a host. ITN exposure altered the observed behaviour of the two susceptible strains, which exhibited a decreased response to the host during ITN tests. The resistant strains did not alter their behaviour during ITN tests. Significantly less net contact was observed during Olyset Net tests compared to Permanet 2.0. The host presence affected survival after exposure: Banfora and VK7 mosquitoes exposed to Permanet 2.0 with a host lived longer compared to tests performed without a host. However, mosquitoes that blood-fed and survived long enough to digest the blood meal did not exhibit significantly reduced longevity regardless of the presence of the host attractant. Conclusions Simple modifications to the WHO cone test and extension of post-test monitoring beyond the current 24 h enable detailed behavioural characterizations of individual ITNs to be compiled. The effects observed from testing with a host and including blood feeding suggest that more representative estimates of true of ITN efficacy are gained with these modifications than when using the current testing protocol.

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

Background Cameroon is considering the implementation of indoor residual spraying (IRS) as a complementary measure to control malaria in the context of high pyrethroid resistance in major malaria vectors. Non-pyrethroid insecticide classes such as organophosphates and carbamates may be utilized in IRS due to widespread pyrethroid resistance. However, the success of this strategy depends on good knowledge of the resistance status of malaria vectors to carbamates and organophosphates. Here, we assessed the susceptibility profile of Anopheles gambiae sensu lato with respect to carbamates and organophosphate and the distribution of the molecular mechanism underlying resistance to these insecticides. Methods Anopheles gambiae s.l. mosquitoes were collected from nine settings across the country and bio-assayed with bendiocarb, propoxur and pirimiphos-methyl. The Ace-1 target-site G119S mutation was genotyped using a TaqMan assay. To investigate the polymorphism in the Ace-1 gene, a region of 924 base pairs in a sequence of the gene was amplified from both live and dead females of An. gambiae exposed to bendiocarb. Results Pirimiphos-methyl induced full mortality in An. gambiae s.l. from all study sites, whereas for carbamates, resistance was observed in four localities, with the lowest mortality rate recorded in Mangoum (17.78 ± 5.02% for bendiocarb and 18.61 ± 3.86% for propoxur) in the southern part of Cameroon. Anopheles coluzzii was found to be the predominant species in the northern tropical part of the country where it is sympatric with Anopheles arabiensis. In the localities situated in southern equatorial regions, this species was predominant in urban settings, while An. gambiae was the most abundant species in rural areas. The G119S Ace-1 target-site mutation was detected only in An. gambiae and only in the sites located in southern Cameroon. Phylogenetic analyses showed a clustering according to the phenotype. Conclusion The occurrence of the Ace-1 target-site substitution G119S in An. gambiae s.l. populations highlights the challenge associated with the impending deployment of IRS in Cameroon using carbamates or organophosphates. It is therefore important to think about a resistance management plan including the use of other insecticide classes such as neonicotinoids or pyrrole to guarantee the implementation of IRS in Cameroon. Graphical Abstract

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

Background Pyrethroid-PBO nets were conditionally recommended for control of malaria transmitted by mosquitoes with oxidase-based pyrethroid-resistance based on epidemiological evidence of additional protective effect with Olyset Plus compared to a pyrethroid-only net (Olyset Net). Entomological studies can be used to assess the comparative performance of other brands of pyrethroid-PBO ITNs to Olyset Plus. Methods An experimental hut trial was performed in Cové, Benin to compare PermaNet 3.0 (deltamethrin plus PBO on roof panel only) to Olyset Plus (permethrin plus PBO on all panels) against wild pyrethroid-resistant Anopheles gambiae sensu lato (s.l.) following World Health Organization (WHO) guidelines. Both nets were tested unwashed and after 20 standardized washes compared to Olyset Net. Laboratory bioassays were also performed to help explain findings in the experimental huts. Results With unwashed nets, mosquito mortality was higher in huts with PermaNet 3.0 compared to Olyset Plus (41% vs. 28%, P Conclusion Olyset Plus outperformed PermaNet 3.0 in terms of its ability to cause greater margins of improved mosquito mortality compared to a standard pyrethroid net, after multiple standardized washes. However, using a margin of non-inferiority defined by the WHO, PermaNet 3.0 was non-inferior to Olyset Plus in inducing mosquito mortality. Considering the low levels of mortality observed and increasing pyrethroid-resistance in West Africa, it is unclear whether either of these nets would demonstrate the same epidemiological impact observed in community trials in East Africa.

Background The practice of agriculture in urban settings contributes to the rapid expansion of insecticide resistance in malaria vectors. However, there is still not enough information on pesticide usage in most urban settings. The present study aims to assess the evolution of Anopheles gambiae (s.l.) population susceptibility to insecticides and patterns of pesticide usage in agriculture in the city of Yaoundé, Cameroon. Methods WHO susceptibility tests and synergist PBO bioassays were conducted on adult An. gambiae (s.l.) mosquitoes aged 3 to 5 days emerging from larvae collected from the field. Seven insecticides (deltamethrin, permethrin, DDT, bendiocarb, propoxur, fenitrothion and malathion) were evaluated. The presence of target site mutation conferring knockdown (kdr) resistance was investigated using TaqMan assay, and mosquito species were identified using SINE-PCR. Surveys on 81 retailers and 232 farmers were conducted to assess general knowledge and practices regarding agricultural pesticide usage. Results High resistance intensity to pyrethroids was observed with a high frequency of the kdr allele 1014F and low frequency of the kdr 1014S allele. The level of susceptibility of An. gambiae (s.l.) to pyrethroids and carbamates was found to decrease with time (from > 34% in 2017 to An. gambiae (s.s.) and An. coluzzii were recorded. Over 150 pesticides and fertilizers were sold by retailers for agricultural purposes in the city of Yaoundé. Most farmers do not respect safety practices. Poor practices including extensive and inappropriate application of pesticides as well as poor management of perished pesticides and empty pesticide containers were also documented. Conclusions The study indicated rapid evolution of insecticide resistance and uncontrolled usage of pesticides by farmers in agriculture. There is an urgent need to address these gaps to improve the management of insecticide resistance. Graphical Abstract

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

Transmission of Plasmodium falciparum malaria parasites occurs when nocturnal Anopheles mosquito vectors feed on human blood. In Africa, where malaria burden is highest, bednets treated with pyrethroid insecticide were highly effective in preventing mosquito bites and reducing transmission, and essential to achieving unprecedented reductions in malaria until 2015 (ref. 1). Since then, progress has stalled2, and with insecticidal bednets losing efficacy against pyrethroid-resistant Anopheles vectors3,4, methods that restore performance are urgently needed to eliminate any risk of malaria returning to the levels seen before their widespread use throughout sub-Saharan Africa5. Here, we show that the primary malaria vector Anopheles gambiae is targeted and killed by small insecticidal net barriers positioned above a standard bednet in a spatial region of high mosquito activity but zero contact with sleepers, opening the way for deploying many more insecticides on bednets than is currently possible. Tested against wild pyrethroid-resistant A. gambiae in Burkina Faso, pyrethroid bednets with organophosphate barriers achieved significantly higher killing rates than bednets alone. Treated barriers on untreated bednets were equally effective, without significant loss of personal protection. Mathematical modelling of transmission dynamics predicted reductions in clinical malaria incidence with barrier bednets that matched those of ‘next-generation’ nets recommended by the World Health Organization against resistant vectors. Mathematical models of mosquito–barrier interactions identified alternative barrier designs to increase performance. Barrier bednets that overcome insecticide resistance are feasible using existing insecticides and production technology, and early implementation of affordable vector control tools is a realistic prospect. The addition of a small net barrier above a standard bednet targets malaria vectors and—as these barriers are further distanced from sleepers—paves the way for the use of a wider range of insecticides to curtail malaria transmission.

BackgroundThe Sahel region of Chad Republic is a prime candidate for malaria pre-elimination. To facilitate pre-elimination efforts in this region, two populations ofAnopheles coluzziifrom Central Chad Republic were characterized, their insecticide resistance profile and the possible molecular mechanisms driving the resistance in the field investigated.MethodsBloodfed femaleAnopheles gambiaes.l. resting indoor, were collected at N’djamena and Massakory, Chad in 2018 and characterized for species composition, and infection rate was determined using the TaqMan assay. Susceptibility to various insecticides was assessed using WHO tube bioassays. Cone bioassays were conducted using various long-lasting insecticidal nets (LLINs). Results were analysed using Chi Square test. Knockdown resistance (kdr) andace-1markers were investigated by TaqMan genotyping.ResultsAnopheles coluzziiwas the major vector found in N’djamena (100%) and Massakory (~ 94%). NoPlasmodiumwas found in 147 bloodfed F0An. coluzzii(82 from N’djamena and 65 from Massakory).High intensity pyrethroid resistance was observed with mortalities of χ2 = 22.8, df = 1,P = 0.0006; mortality = 13.0% for deltamethrin,χ2 = 8.8, df = 1,P ace-1mutation points to the role of metabolic resistance in the bendiocarb resistance. Both 1014F and 1014S mutations were found in both populations at around 60% and ConclusionsMultiple resistance inAn. coluzziipopulations from Chad highlight challenges associated with deployment of LLINs and indoor residual spraying (IRS) in the Sahel of this country. The pyrethroid-synergists LLINs (e.g. PermaNet®3.0) and organophosphate-based IRS maybe the alternatives for malaria control in this region.

BackgroundMonitoring of drug resistance in Plasmodium populations is crucial for malaria control. This has primarily been performed in humans and rarely in mosquitoes where parasites genetic recombination occurs. Here, we characterized the Plasmodium spp populations in wild Anopheles vectors by analyzing the genetic diversity of the P. falciparum kelch13 and mdr1 gene fragments implicated in artemisinin and partner drug resistance across Cameroon in three major malaria vectors.MethodsAnopheles mosquitoes were collected across nine localities in Cameroon and dissected into the head/thorax (H/T) and abdomen (Abd) after species identification. A TaqMan assay was performed to detect Plasmodium infection. Fragments of the Kelch 13 and mdr1 genes were amplified in P. falciparum positive samples and directly sequenced to assess their drug resistance polymorphisms and genetic diversity profile.ResultsThe study revealed a high Plasmodium infection rate in the major Anopheles vectors across Cameroon. Notably, An. funestus vector recorded the highest sporozoite (8.02%) and oocyst (14.41%) infection rates. A high P. falciparum sporozoite rate (80.08%) alongside epidemiological signatures of significant P. malariae (15.94%) circulation were recorded in these vectors. Low genetic diversity with six (A578S, R575I, G450R, L663L, G453D, N458D) and eight (H53H, V62L, V77E, N86Y, G102G, L132I, H143H, Y184F) point mutations were observed in the k13 and mdr1 backbones respectively. Remarkably, the R575I (4.44%) k13 and Y184F (64.2%) mdr1 mutations were the predominant variants in the P. falciparum populations.ConclusionThe emerging signal of the R575I polymorphism in the Pfk13 propeller backbone entails the regular surveillance of molecular markers to inform evidence-based policy decisions. Moreover, the high frequency of the 86N184F allele highlights concerns on the plausible decline in efficacy of artemisinin-combination therapies (ACTs); further implying that parasite genotyping from mosquitoes can provide a more relevant scale for quantifying resistance epidemiology in the field.

The mutualistic association between Wolbachia endosymbionts and their filarial nematode hosts has been exploited as a validated drug target delivering macrofilaricidal outcomes. Limitations of existing antibiotics to scale-up have driven the search for new drugs, which are effective in shorter regimens of 7 days or less. Here, we review the last 14 years of anti-Wolbachia drug discovery by the anti-Wolbachia (A·WOL) consortium, which has screened more than two million compounds, delivering thousands of hit compounds. Refined screening models integrated with robust pharmacokinetic/pharmacodynamic (PK/PD) driven optimisation and selection strategies have delivered the first two drug candidates specifically designed to target Wolbachia. AWZ1066S and ABBV-4083 are currently progressing through clinical trials with the aim of delivering safe and effective macrofilaricides to support the elimination of onchocerciasis and lymphatic filariasis.

Background In common with many South East Asian countries, Malaysia is endemic for dengue. Dengue control in Malaysia is currently based on reactive vector management within 24 h of a dengue case being reported. Preventive rather than reactive vector control approaches, with combined interventions, are expected to improve the cost-effectiveness of dengue control programs. The principal objective of this cluster randomized controlled trial is to quantify the effectiveness of a preventive integrated vector management (IVM) strategy on the incidence of dengue as compared to routine vector control efforts. Methods The trial is conducted in randomly allocated clusters of low- and medium-cost housing located in the Federal Territory of Kuala Lumpur and Putrajaya. The IVM approach combines: targeted outdoor residual spraying with K-Othrine Polyzone, deployment of mosquito traps as auto-dissemination devices, and community engagement activities. The trial includes 300 clusters randomly allocated in a 1:1 ratio. The clusters receive either the preventive IVM in addition to the routine vector control activities or the routine vector control activities only. Epidemiological data from monthly confirmed dengue cases during the study period will be obtained from the Vector Borne Disease Sector, Malaysian Ministry of Health e-Dengue surveillance system. Entomological surveillance data will be collected in 12 clusters randomly selected from each arm. To measure the effectiveness of the IVM approach on dengue incidence, a negative binomial regression model will be used to compare the incidence between control and intervention clusters. To quantify the effect of the interventions on the main entomological outcome, ovitrap index, a modified ordinary least squares regression model using a robust standard error estimator will be used. Discussion Considering the ongoing expansion of dengue burden in Malaysia, setting up proactive control strategies is critical. Despite some limitations of the trial such as the use of passive surveillance to identify cases, the results will be informative for a better understanding of effectiveness of proactive IVM approach in the control of dengue. Evidence from this trial may help justify investment in preventive IVM approaches as preferred to reactive case management strategies. Trial registration ISRCTN ISRCTN81915073. Retrospectively registered on 17 April 2020.

BACKGROUND\ud Pyrethroids are the most widely used insecticides for the control of malaria transmitting Anopheles gambiae mosquitoes and rapid increase in resistance to this insecticide class is of major concern. Pyrethroids target the Voltage Gated Sodium Channels (VGSCs), that have a key role in the normal function of the mosquitoes' nervous system. VGSC mutations L995F and L995S have long been associated with pyrethroid resistance and screening for their presence is routine in insecticide resistance management programs. Recently, a VGSC haplotype containing two amino acid substitutions associated with resistance in other species, V402L and I1527T, was identified. These two VGSC mutations are found in tight linkage and are mutually exclusive to the classical L995F/S mutations.\ud \ud RESULTS\ud We identify the presence of the V402L-I1527 T haplotype in resistant An. coluzzii colonized strains and in field populations from Burkina Faso at frequencies higher than previously reported; in some cases almost reaching fixation. Functional validation of V402L in insecticide resistance using a CRISPR/Cas9 genome modified line showed that it confers reduced mortality after exposure to all tested pyrethroids and DDT, but at lower levels compared to L995F. In contrast however to L995F, no fitness costs were identified for mosquitoes carrying V402L under laboratory conditions.\ud \ud CONCLUSION\ud The V402L substitution confers pyrethroid resistance in An. gambiae in the absence of any other VGSC substitution and/or alternative resistance mechanisms. The lower fitness cost associated with this kdr mutation may provide a selective advantage over the classical kdr in some settings and genotyping at this locus should be added in the list of resistant alleles for routine screening.

Background Insecticidal mosquito vector control products are vital components of malaria control programmes. Test facilities are key in assessing the effectiveness of vector control products against local mosquito populations, in environments where they will be used. Data from these test facilities must be of a high quality to be accepted by regulatory authorities, including the WHO Prequalification Team for vector control products. In 2013–4, seven insecticide testing facilities across sub-Saharan Africa, with technical and financial support from Innovative Vector Control Consortium (IVCC), began development and implementation of quality management system compliant with the principles of Good Laboratory Practice (GLP) to improve data quality and reliability. Methods and principle findings We conducted semi-structured interviews, emails, and video-call interviews with individuals at five test facilities engaged in the IVCC-supported programme and working towards or having achieved GLP. We used framework analysis to identify and describe factors affeting progress towards GLP. We found that eight factors were instrumental in progress, and that test facilities had varying levels of control over these factors. They had high control over the training programme, project planning, and senior leadership support; medium control over infrastructure development, staff structure, and procurement; and low control over funding the availability and accessibility of relevant expertise. Collaboration with IVCC and other partners was key to overcoming the challenges associated with low and medium control factors. Conclusion For partnership and consortia models of research capacity strengthening, test facilities can use their own internal resources to address identified high-control factors. Project plans should allow additional time for interaction with external agencies to address medium-control factors, and partners with access to expertise and funding should concentrate their efforts on supporting institutions to address low-control factors. In practice, this includes planning for financial sustainability at the outset, and acting to strengthen national and regional training capacity.

Climate change lies at the heart of many complex humanitarian emergencies and emerging global health challenges, with Sub-Saharan Africa (SSA) identified as one of the most vulnerable regions to the impacts of climate change.1 An arc stretching from Somalia and Eritrea in the east to Mauritania in the west forms a band of countries that are particularly vulnerable to the consequences of climate change (Figure 1).2 This arc experiences a devastating combination of state fragmentation, Islamist insurgency, and climate change, undermining livelihood strategies across the region. A climate injustice exists; despite contributing relatively little to the anthropogenic causes of climate change, individuals living in these countries face the most severe impacts

Background Vector control is the main intervention used to control arboviral diseases transmitted by Aedes mosquitoes because there are no effective vaccines or treatments for most of them. Control of Aedes mosquitoes relies heavily on the use of insecticides, the effectiveness of which may be impacted by resistance. In addition, rational insecticide application requires detailed knowledge of vector distribution, dynamics, resting, and feeding behaviours, which are poorly understood for Aedes mosquitoes in Africa. This study investigated the spatiotemporal distribution and insecticide resistance status of Aedes aegypti across ecological extremes of Ghana. Methods Immature mosquitoes were sampled from containers in and around human dwellings at seven study sites in urban, suburban, and rural areas of Ghana. Adult Aedes mosquitoes were sampled indoors and outdoors using Biogents BG-Sentinel 2 mosquito traps, human landing catches, and Prokopack aspiration. Distributions of immature and adult Aedes mosquitoes were determined indoors and outdoors during dry and rainy seasons at all sites. The phenotypic resistance status of Aedes mosquitoes to insecticides was determined using World Health Organization susceptibility bioassays. The host blood meal source was determined by polymerase chain reaction. Results A total of 16,711 immature Aedes were sampled, with over 70% found in car tyres. Significantly more breeding containers had Aedes immatures during the rainy season (11,856; 70.95%) compared to the dry season (4855; 29.05%). A total of 1895 adult Aedes mosquitos were collected, including Aedes aegypti (97.8%), Aedes africanus (2.1%) and Aedesluteocephalus (0.1%). Indoor sampling of adult Aedes yielded a total of 381 (20.1%) and outdoor sampling a total of 1514 (79.9%) mosquitoes (z = − 5.427, P = 0.0000) over the entire sampling period. Aedes aegypti populations were resistant to dichlorodiphenyltrichloroethane at all study sites. Vectors showed suspected resistance to bendiocarb (96–97%), permethrin (90–96%) and deltamethrin (91–96%), and were susceptible to the organophosphate for all study sites. Blood meal analysis showed that the Aedes mosquitoes were mostly anthropophilic, with a human blood index of 0.9 (i.e. humans, 90%; human and dog, 5%; dog and cow, 5%). Conclusions Aedes mosquitoes were found at high densities in all ecological zones of Ghana. Resistance of Aedes spp. to pyrethroids and carbamates may limit the efficacy of vector control programmes and thus requires careful monitoring. Graphical abstract

Background The excessive use of synthetic insecticides is responsible for many cases of resistance in insects. Therefore, the use of natural molecules of ecological interest with insecticidal properties is an alternative approach to the use of synthetic insecticides. The aim of this study is to investigating the larvicidal and adulticidal activity and the chemical composition of the essential oil of Aeollanthus pubescens on the major malaria vector, Anopheles gambiae. Methods Three reference strains of Anopheles gambiae sensu stricto (Kisumu, Kiskdr and Acerkis) were used in this study. The leaves of A. pubescens were collected in southern Benin. The standard World Health Organisation (WHO) guidelines for larvicide evaluation were used, and the chemical composition of the essential oil was analysed by gas chromatography coupled to mass spectrometry. Adult mosquitoes of each strain were exposed to pieces of net coated with the essential oil for 3 min using the WHO cone bioassay method. Probit regression analysis was used to determine the concentrations that would kill 50 and 95% of each test population (LC50, LC95) and the knockdown time for 50 and 95% of each test population (KDT50, and KDT95). The difference between the mortality–dose regressions for the different strains was analysed using the likelihood ratio test (LRT). The log-rank test was performed to evaluate the difference in survival between the strains. Results A total of 14 components were identified, accounting for 98.3% of total oil content. The major components were carvacrol (51.1%), thymyle acetate (14.0%) and ɣ-terpinene (10.6%). The essential oil showed larvicidal properties on the Kisumu, Acerkis and Kiskdr strains, with LC50 of 29.6, 22.9 and 28.4 ppm, respectively. With pieces of netting treated at 165 µg/cm2, the KDT50 of both Acerkis (1.71 s; Z = 3.34, P Z = 3.49, P χ2 = 99, df = 1, P χ2 = 117, df = 1, P χ2 = 96.9, df = 1, P Conclusion Our findings show that A. pubescens essential oil has larvicide and adulticide properties against the malaria vector An. gambiae sensu stricto, suggesting that this essential oil may be a potential candidate for the control of the resistant malaria-transmitting vectors. Graphical Abstract

Background Insecticide-treated bed nets and indoor residual spraying comprise the major control measures against Anopheles gambiae sl, the dominant vector in sub-Saharan Africa. The primary site of contact with insecticide is through the mosquitoes’ legs, which represents the first barrier insecticides have to bypass to reach their neuronal targets. Proteomic changes and leg cuticle modifications have been associated with insecticide resistance that may reduce the rate of penetration of insecticides. Here, we performed a multiple transcriptomic analyses focusing on An. coluzzii legs. Results Firstly, leg-specific enrichment analysis identified 359 genes including the pyrethroid-binder SAP2 and 2 other chemosensory proteins, along with 4 ABCG transporters previously shown to be leg enriched. Enrichment of gene families included those involved in detecting chemical stimuli, including gustatory and ionotropic receptors and genes implicated in hydrocarbon-synthesis. Subsequently, we compared transcript expression in the legs of a highly resistant strain (VK7-HR) to both a strain with very similar genetic background which has reverted to susceptibility after several generations without insecticide pressure (VK7-LR) and a lab susceptible population (NG). Two hundred thirty-two differentially expressed genes (73 up-regulated and 159 down-regulated) were identified in the resistant strain when compared to the two susceptible counterparts, indicating an over-expression of phase I detoxification enzymes and cuticular proteins, with decrease in hormone-related metabolic processes in legs from the insecticide resistant population. Finally, we analysed the short-term effect of pyrethroid exposure on An. coluzzii legs, comparing legs of 1 h-deltamethrin-exposed An. coluzzii (VK7-IN) to those of unexposed mosquitoes (VK7-HR) and identified 348 up-regulated genes including those encoding for GPCRs, ABC transporters, odorant-binding proteins and members of the divergent salivary gland protein family. Conclusions The data on An. coluzzii leg-specific transcriptome provides valuable insights into the first line of defense in pyrethroid resistant and short-term deltamethrin-exposed mosquitoes. Our results suggest that xenobiotic detoxification is likely occurring in legs, while the enrichment of sensory proteins, ABCG transporters and cuticular genes is also evident. Constitutive resistance is primarily associated with elevated levels of detoxification and cuticular genes, while short-term insecticide-induced tolerance is linked with overexpression of transporters, GPCRs and GPCR-related genes, sensory/binding and salivary gland proteins.

Simple Summary A group of insecticides, called pyrethroids, are the main strategy for controlling the mosquito vectors of malaria. Pyrethroids are used in all insecticide-treated bednets, and many indoor residual spray programmes (in which insecticides are sprayed on the interior walls of houses). There are different types of pyrethroids within the class (e.g., deltamethrin and permethrin). Across the world, mosquitoes are showing signs of resistance to the pyrethroids, such as reduced mortality following contact. However, it is unclear if this resistance is uniform across the pyrethroid class (i.e., if a mosquito is resistant to deltamethrin, whether it is resistant to permethrin at the same level). In addition, it is not known if switching between different pyrethroids can be used to effectively maintain mosquito control when resistance to a single pyrethroid has been detected. This review examined the evidence from molecular studies, resistance testing from laboratory and field data, and mosquito behavioural assays to answer these questions. The evidence suggested that in areas where pyrethroid resistance exists, different mortality seen between the pyrethroids is not necessarily indicative of an operationally relevant difference in control performance, and there is no reason to rotate between common pyrethroids (i.e., deltamethrin, permethrin, and alpha-cypermethrin) as an insecticide resistance management strategy. Abstract Pyrethroid resistance is widespread in malaria vectors. However, differential mortality in discriminating dose assays to different pyrethroids is often observed in wild populations. When this occurs, it is unclear if this differential mortality should be interpreted as an indication of differential levels of susceptibility within the pyrethroid class, and if so, if countries should consider selecting one specific pyrethroid for programmatic use over another. A review of evidence from molecular studies, resistance testing with laboratory colonies and wild populations, and mosquito behavioural assays were conducted to answer these questions. Evidence suggested that in areas where pyrethroid resistance exists, different results in insecticide susceptibility assays with specific pyrethroids currently in common use (deltamethrin, permethrin, α-cypermethrin, and λ-cyhalothrin) are not necessarily indicative of an operationally relevant difference in potential performance. Consequently, it is not advisable to use rotation between these pyrethroids as an insecticide-resistance management strategy. Less commonly used pyrethroids (bifenthrin and etofenprox) may have sufficiently different modes of action, though further work is needed to examine how this may apply to insecticide resistance management.

Insecticide resistance in Anopheles mosquitoes is a major obstacle in maintaining the momentum in reducing the malaria burden; mitigating strategies require improved understanding of the underlying mechanisms. Mutations in the target site of insecticides (the voltage gated sodium channel for the most widely used pyrethroid class) and over-expression of detoxification enzymes are commonly reported, but their relative contribution to phenotypic resistance remain poorly understood. Here we present a genome editing pipeline to introduce single nucleotide polymorphisms in An. gambiae which we have used to study the effect of the classical kdr mutation L1014F (L995F based on An. gambiae numbering), one of the most widely distributed resistance alleles. Introduction of 1014F in an otherwise fully susceptible genetic background increased levels of resistance to all tested pyrethroids and DDT ranging from 9.9-fold for permethrin to >24-fold for DDT. The introduction of the 1014F allele was sufficient to reduce mortality of mosquitoes after exposure to deltamethrin treated bednets, even as the only resistance mechanism present. When 1014F was combined with over-expression of glutathione transferase Gste2, resistance to permethrin increased further demonstrating the critical combined effect between target site resistance and detoxification enzymes in vivo. We also show that mosquitoes carrying the 1014F allele in homozygosity showed fitness disadvantages including increased mortality at the larval stage and a reduction in fecundity and adult longevity, which can have consequences for the strength of selection that will apply to this allele in the field., Author summary Escalation of pyrethroid resistance in Anopheles mosquitoes threatens to reduce the effectiveness of our most important tools in malaria control. Studying the mechanisms underlying insecticide resistance is critical to design mitigation strategies. Here, using genome modified mosquitoes, we functionally characterize the most prevalent mutation in resistant mosquitoes, showing that it confers substantial levels of resistance to all tested pyrethroids and undermines the performance of pyrethroid-treated nets. Furthermore, we show that combining this mutation with elevated levels of a detoxification enzyme further increases resistance. The pipeline we have developed provides a robust approach to quantifying the contribution of different combinations of resistance mechanisms to the overall phenotype, providing the missing link between resistance monitoring and predictions of resistance impact.

The COVID-19 pandemic is currently one of the major global health and economic challenges. An efficient method for reducing the transmission of the virus is a still unmet medical need. Existing experimental data have shown that coronavirus survival is negatively impacted by ozone, high temperature, and low humidity. Therefore, it is feasible to use area ozonation in pharmacies - the front line of the healthcare system. Nevertheless, further work is needed to evaluate the effectiveness of ozone disinfection to reduce the transmission of this virus in pharmacies, hospitals, and other public environments. Med Pr. 2021;72(5):529-34.

Background\ud Indoor residual spraying (IRS) of insecticides is a key method to reduce vector transmission of Trypanosoma cruzi, causing Chagas disease in a large part of South America. However, the successes of IRS in the Gran Chaco region straddling Bolivia, Argentina, and Paraguay, have not equalled those in other Southern Cone countries.\ud Aims\ud This study evaluated routine IRS practices and insecticide quality control in a typical endemic community in the Bolivian Chaco.\ud Methods\ud Alpha-cypermethrin active ingredient (a.i.) captured onto filter papers fitted to sprayed wall surfaces, and in prepared spray tank solutions, were measured using an adapted Insecticide Quantification Kit (IQK™) validated against HPLC quantification methods. The data were analysed by mixed-effects negative binomial regression models to examine the delivered insecticide a.i. concentrations on filter papers in relation to the sprayed wall heights, spray coverage rates (surface area / spray time [m2/min]), and observed/expected spray rate ratios. Variations between health workers and householders’ compliance to empty houses for IRS delivery were also evaluated. Sedimentation rates of alpha-cypermethrin a.i. post-mixing of prepared spray tanks were quantified in the laboratory.\ud \ud Results\ud Substantial variations were observed in the alpha-cypermethrin a.i. concentrations delivered; only 10.4% (50/480) of filter papers and 8.8% (5/57) of houses received the target concentration of 50 mg ± 20% a.i./m2. The delivered concentrations were not related to those in the matched spray tank solutions. The sedimentation of alpha-cypermethrin a.i. in the surface solution of prepared spray tanks was rapid post-mixing, resulting in a linear 3.3% loss of a.i. content per minute and 49% loss after 15 min. Only 7.5% (6/80) of houses were sprayed at the WHO recommended rate of 19 m2/min (± 10%), whereas 77.5% (62/80) were sprayed at a lower than expected rate. The median a.i. concentration delivered to houses was not significantly associated with the observed spray coverage rate. Householder compliance did not significantly influence either the spray coverage rates or the median alpha-cypermethrin a.i. concentrations delivered to houses.\ud Conclusions\ud Suboptimal delivery of IRS is partially attributable to the insecticide physical characteristics and the need for revision of insecticide delivery methods, which includes training of IRS teams and community education to encourage compliance. The IQK™ is a necessary field-friendly tool to improve IRS quality and to facilitate health worker training and decision-making by Chagas disease vector control managers.

Background Following earlier trials indicating that their potential in dengue vector control was constrained by housing structure, a large-scale cluster-randomized trial of insecticide treated curtains (ITCs) and water jar covers (ITJCs) was undertaken in Venezuela. Methods In Trujillo, Venezuela, 60 clusters (6223 houses total) were randomized so that 15 clusters each received either PermaNet insecticide-treated window curtains (ITCs), permanent insecticide-treated water storage jar covers (ITJCs), a combination of both ITCs and ITJCs, or no insecticide treated materials (ITMs). A further 15 clusters located at least 5km from the edge of the study site were selected to act as an external control. Entomological surveys were carried out immediately before and after intervention, and then at 6-month intervals over the following 27 months. The Breteau and House indices were used as primary outcome measures and ovitrap indices as secondary. Negative binomial regression models were used to compare cluster-level values of these indices between the trial arms. Results Reductions in entomological indices followed deployment of all ITMs and throughout the trial, indices in the external control arm remained substantially higher than in the ITM study arms including the internal control. Comparing the ratios of between-arm means to summarise the entomological indices throughout the study, the combined ITC+ITJC intervention had the greatest impact on the indices, with a 63% difference in the pupae per person indices between the ITC+ITJC arm and the internal control. However, coverage had fallen below 60% by 14-months post-intervention and remained below 40% for most of the remaining study period. Conclusions ITMs can impact dengue vector populations in the long term, particularly when ITCs and ITJCs are deployed in combination. Trial registration ClinicalTrials.gov ISRCTN08474420; www.isrctn.com.

Pyrethroid-impregnated nets have contributed significantly to halving the burden of malaria but resistance threatens their future efficacy and the pipeline of new insecticides is short. Here we report that an invertebrate automated phenotyping platform (INVAPP), combined with the algorithm Paragon, provides a robust system for measuring larval motility in Anopheles gambiae (and An. coluzzi) as well as Aedes aegypti with the capacity for high-throughput screening for new larvicides. By this means, we reliably quantified both time- and concentration-dependent actions of chemical insecticides faster than using the WHO standard larval assay. We illustrate the effectiveness of the system using an established larvicide (temephos) and demonstrate its capacity for library-scale chemical screening using the Medicines for Malaria Venture (MMV) Pathogen Box library. As a proof-of-principle, this library screen identified a compound, subsequently confirmed to be tolfenpyrad, as an effective larvicide. We have also used the INVAPP / Paragon system to compare responses in larvae derived from WHO classified deltamethrin resistant and sensitive mosquitoes. We show how this approach to monitoring larval response to insecticides can be adapted for use with a smartphone camera application and therefore has potential for further development as a simple portable field-assay with associated real-time, geo-located information to identify hotspots.

Background\ud Alcohol-based hand rub (ABHR) is widely used in both health and social facilities to prevent infection, but it is not known whether supplying it for regular perinatal use can prevent newborn sepsis in African rural homes. Our study piloted a cluster randomised trial of providing ABHR to postpartum mothers to prevent neonatal infection-related morbidity in the communities.\ud Methods\ud We conducted a pilot parallel cluster randomised controlled trial across ten villages (clusters) in rural Eastern Uganda. Pregnant women of over 34 weeks' gestation were recruited over a period of 3 months. Both clusters received the standard of care of antenatal health education, Maama Kit, and clinic appointments. In addition, women in the intervention villages received ABHR, instructions on ABHR use, a poster on the 'three moments of hand hygiene', and training. We followed up each mother-baby pair for 3 months after birth and measured rates of consent, recruitment, and follow-up (our target rate was more than 80%). Other measures included ABHR use (the acceptable use was more than four times a day) and its mode of distribution (village health workers (VHWs) or pharmacy), acceptability of study protocol and electronic data capture, and the use of WHO Integrated Management of Childhood Illness (IMCI) tool to screen for newborn infection.\ud Results\ud We selected 36% (10/28) of villages for randomisation to either intervention or control. Over 12 weeks, 176 pregnant women were screened and 58.5% (103/176) were eligible. All, 100% (103/103), eligible women gave consent and were enrolled into the trial (55 intervention and 48 control). After birth, 94.5% (52/55) of mothers in the intervention and 100% (48/48) of mothers in the control villages were followed up within 72 h. Most, 90.9% (50/55), of the mothers in the intervention villages (96.2% of live births) and 95.8% (46/48) of mothers in the control villages (95.9% of live births) were followed up at 3 months. In intervention villages, the average hand rub use was 6.6 times per day. VHWs accounted for all ABHR stock, compared to the pharmacy that could not account for 5 l of ABHR. The screening tool was positive for infection among a third of babies, i.e. 29.2% (14/48) in the intervention villages versus 31.4% (16/51) in the control villages.VHWs completed the first four questions of IMCI screening tool with ease and accuracy. There were no adverse reactions with the ABHR.\ud Conclusion\ud It is feasible to conduct a cluster-randomised controlled trial (cRCT) of the provision of ABHR to postpartum mothers to prevent neonatal infection-related morbidity in the community in resource-poor settings. Our results indicate that home recruitment promotes excellent follow-up and retention of participants in community trials. The intervention was safe. This pilot study informed the substantial changes necessary in the larger cRCT, including a change in the primary outcome to a composite outcome considering multiple methods of infection detection. A large BabyGel cluster randomised controlled trial is now required.\ud \ud Trial registration\ud \ud ISRCTN67852437, registered March 02, 2015.\ud \ud Trial funding\ud \ud Medical Research Council/WellcomeTrust/DfID (Global Health Trials Scheme).

Until an effective vaccine against SARS-CoV-2 is available on a widespread scale, the control of the COVID-19 pandemic is\ud reliant upon effective pandemic control measures. The ability of SARS-CoV-2 to remain viable on surfaces and in aerosols,\ud means indirect contact transmission can occur and there is an opportunity to reduce transmission using effective disinfectants\ud in public and communal spaces. Virusend (TX-10), a novel disinfectant, has been developed as a highly effective disinfectant\ud against a range of microbial agents. Here we investigate the ability of Virusend to inactivate SARS-CoV-2. Using surface and solution inactivation assays, we show that Virusend is able to reduce SARS-CoV-2 viral titre by 4 log10 p.f.u. ml−1 within 1min of contact. Ensuring disinfectants are highly effective against SARS-CoV-2 is important in eliminating environmental sources of the virus to control the COVID-19 pandemic

OBJECTIVES \ud The control of the COVID-19 pandemic in the UK has necessitated restrictions on amateur and professional sports due to the perceived infection risk to competitors, via direct person to person transmission, or possibly via the surfaces of sports equipment. The sharing of sports equipment such as tennis balls was therefore banned by some sport’s governing bodies. We sought to investigate the potential of sporting equipment as transmission vectors of SARS-CoV-2.\ud Methods\ud Ten different types of sporting equipment, including balls from common sports, were inoculated with 40μl droplets containing clinically relevant concentrations of live SARS-CoV-2 virus. Materials were then swabbed at time points relevant to sports (1, 5, 15, 30, 90 minutes). The amount of live SARS-CoV-2 recovered at each time point was enumerated using viral plaque assays, and viral decay and half-life was estimated through fitting linear models to log transformed data from each material. \ud RESULTS\ud At one minute, SARS-CoV-2 virus was recovered in only seven of the ten types of equipment with the low dose inoculum, one at five minutes and none at 15 minutes. Retrievable virus dropped significantly for all materials tested using the high dose inoculum with mean recovery of virus falling to 0.74% at 1 minute, 0.39% at 15 minutes and 0.003% at 90 minutes. Viral recovery, predicted decay, and half-life varied between materials with porous surfaces limiting virus transmission. \ud CONCLUSIONS \ud This study shows that there is an exponential reduction in SARS-CoV-2 recoverable from a range of sports equipment after a short time period, and virus is less transferrable from materials such as a tennis ball, red cricket ball and cricket glove. Given this rapid loss of viral load and the fact that transmission requires a significant inoculum to be transferred from equipment to the mucous membranes of another individual it seems unlikely that sports equipment is a major cause for transmission of SARS-CoV-2. These findings have important policy implications in the context of the pandemic and may promote other infection control measures in sports to reduce the risk of SARS-CoV-2 transmission and urge sports equipment manufacturers to identify surfaces that may or may not be likely to retain transferable virus.

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.

Varroa destructor is one of the main problems in modern beekeeping. Highly selective acaricides with low toxicity to bees are used internationally to control this mite. One of the key acaricides is the organophosphorus (OP) proinsecticide coumaphos, that becomes toxic after enzymatic activation inside Varroa. We show here that mites from the island Andros (AN-CR) exhibit high levels of coumaphos resistance. Resistance is not mediated by decreased coumaphos uptake, target-site resistance, or increased detoxification. Reduced proinsecticide activation by a cytochrome P450 enzyme was the main resistance mechanism, a powerful and rarely encountered evolutionary solution to insecticide selection pressure. After treatment with sublethal doses of [(14)C] coumaphos, susceptible mite extracts had substantial amounts of coroxon, the activated metabolite of coumaphos, while resistant mites had only trace amounts. This indicates a suppression of the P450 (CYP)-mediated activation step in the AN-CR mites. Bioassays with coroxon to bypass the activation step showed that resistance was dramatically reduced. There are 26 CYPs present in the V. destructor genome. Transcriptome analysis revealed overexpression in resistant mites of CYP4DP24 and underexpression of CYP3012A6 and CYP4EP4. RNA interference of CYP4EP4 in the susceptible population, to mimic underexpression seen in the resistant mites, prevented coumaphos activation and decreased coumaphos toxicity.

Background It is important to understand whether the potential impact of pyrethroid resistance on malaria control can be mitigated by switching between different pyrethroids or whether cross-resistance within this insecticide class precludes this approach. Methods Here we assess the relationships among pyrethroids in terms of their binding affinity to, and depletion by, key cytochrome P450 enzymes (hereafter P450s) that are known to confer metabolic pyrethroid resistance in Anopheles gambiae (s.l.) and An. funestus, in order to identify which pyrethroids may diverge from the others in their vulnerability to resistance. We then investigate whether these same pyrethroids also diverge from the others in terms of resistance in vector populations. Results We found that the type I and II pyrethroids permethrin and deltamethrin, respectively, are closely related in terms of binding affinity to key P450s, depletion by P450s and resistance within vector populations. Bifenthrin, which lacks the common structural moiety of most pyrethroids, diverged from the other pyrethroids tested in terms of both binding affinity to key P450s and depletion by P450s, but resistance to bifenthrin has rarely been tested in vector populations and was not analysed here. Etofenprox, which also lacks the common structural moiety of most pyrethroids, diverged from the more commonly deployed pyrethroids in terms of binding affinity to key P450s and resistance in vector populations, but did not diverge from these pyrethroids in terms of depletion by the P450s. The analysis of depletion by the P450s indicated that etofenprox may be more vulnerable to metabolic resistance mechanisms in vector populations. In addition, greater resistance to etofenprox was found across Aedes aegypti populations, but greater resistance to this compound was not found in any of the malaria vector species analysed. The results for pyrethroid depletion by anopheline P450s in the laboratory were largely not repeated in the findings for resistance in malaria vector populations. Conclusion Importantly, the prevalence of resistance to the pyrethroids α-cypermethrin, cyfluthrin, deltamethrin, λ-cyhalothrin and permethrin was correlated across malaria vector populations, and switching between these compounds as a tool to mitigate against pyrethroid resistance is not advised without strong evidence supporting a true difference in resistance. Graphical Abstract

Vector population control using insecticides is a key element of current strategies to prevent malaria transmission in Africa. The introduction of effective insecticides, such as the organophosphate pirimiphos-methyl, is essential to overcome the recurrent emergence of resistance driven by the highly diverse Anopheles genomes. Here, we use a population genomic approach to investigate the basis of pirimiphos-methyl resistance in the major malaria vectors Anopheles gambiae and A. coluzzii. A combination of copy number variation and a single non-synonymous substitution in the acetylcholinesterase gene, Ace1, provides the key resistance diagnostic in an A. coluzzii population from Côte d’Ivoire that we used for sequence-based association mapping, with replication in other West African populations. The Ace1 substitution and duplications occur on a unique resistance haplotype that evolved in A. gambiae and\ud introgressed into A. coluzzii, and is now common in West Africa primarily due to selection imposed by other organophosphate or carbamate insecticides. Our findings highlight the predictive value of this\ud complex resistance haplotype for phenotypic resistance and clarify its evolutionary history, providing tools to for molecular surveillance of the current and future effectiveness of pirimiphos-methyl based\ud interventions.

Background The efficacy of insecticide-treated nets (ITNs) containing the insect growth regulator pyriproxyfen (PPF) and pyrethroid insecticides (PPF-ITNs) is being assessed in clinical trials to determine whether they provide greater protection from malaria than standard pyrethroid-treated ITNs in areas where mosquitoes are resistant to pyrethroids. Understanding the entomological mode of action of this new ITN class will aide interpretation of the results from these trials. Methods Anopheles gambiae sensu lato (s.l.) mosquitoes from a susceptible laboratory strain were exposed to PPF-treated netting 24 h, 6 h, and immediately prior to, or 24 h post blood feeding, and the impact on fecundity, fertility and longevity recorded. Pyrethroid-resistant populations were exposed to nets containing permethrin and PPF (PPF-ITNs) in cone bioassays and daily mortality recorded. Mosquitoes were also collected from inside houses pre- and post-distribution of PPF-ITNs in a clinical trial conduced in Burkina Faso; female An. gambiae s.l. were then assessed for fecundity and fertility. Results PPF exposure reduced the median adult lifespan of insecticide-susceptible mosquitoes by 4 to 5 days in all exposure times (p An. gambiae s.l. females were collected from five villages between 1 and 12 months before distribution of PPF-ITNs. Of these mosquitoes, 75% laid eggs and the remaining 25% appeared to have normal ovaries upon dissection. In contrast, only 8.6% of the 631 blood-fed or gravid An. gambiae s.l. collected post PPF-ITN distribution successfully oviposited; 276 (43.7%) did not oviposit but had apparently normal ovaries upon dissection, and 301 (47.7%) did not oviposit and had abnormal eggs upon dissection. Egg numbers were also significantly lower (average of 138/female prior distribution vs 85 post distribution, p Conclusion Exposure to a mixture of PPF and pyrethroids on netting shortens the lifespan of mosquitoes and reduces reproductive output. Sterilization of vectors lasted at least one year under operational conditions. These findings suggest a longer effective lifespan of PPF-pyrethroid nets than reported previously.