Resistance development in insects significantly threatens the important benefits obtained by insecticide usage in vector control of disease-transmitting insects. Discovery of new chemical entities with insecticidal activity is highly desired in order to develop new insecticide candidates. Here, we present the design, synthesis, and biological evaluation of phenoxyacetamide-based inhibitors of the essential enzyme acetylcholinesterase 1 (AChE1). AChE1 is a validated insecticide target to control mosquito vectors of, e.g., malaria, dengue, and Zika virus infections. The inhibitors combine a mosquito versus human AChE selectivity with a high potency also for the resistance-conferring mutation G122S; two properties that have proven challenging to combine in a single compound. Structure−activity relationship analyses and molecular dynamics simulations of inhibitor−protein complexes have provided insights that elucidate the molecular basis for these properties. We also show that the inhibitors demonstrate in vivo insecticidal activity on disease-transmitting mosquitoes. Our findings support the concept of noncovalent, selective, and resistance-breaking inhibitors of AChE1 as a promising approach for future insecticide development.

The status of resistance was investigated in Anopheles gambiae sensu lato and An. funestus (Diptera: Culicidae) mosquitoes from western Kenya to four classes of insecticides approved by World Health Organization for indoor residual spraying. The prevalence of the knockdown-resistance (kdr) mutation associated with resistance to pyrethroids and DDT was determined in An. gambiae s.l.. Standard World Health Organization diagnostic bioassay kits for DDT (an organochlorine), fenitrothion (an organophosphate), bendiocarb (a carbamate), and the pyrethoirds, lambdacyhalothrin and permethrin, were used. Knockdown every 10 min and mortality 24 h after exposure were noted. Controls not treated with insecticides and with the susceptible An. gambiae KISUMU strain were included in the bioassays. The presence of the kdr gene was determined using a standard diagnostic polymerase chain reaction assay. Over 98% mortality was observed for tests with all insecticides for both An. gambiae s.l. and An. funestus. Knockdown rates were not significantly different between An. gambiae s.l. and the KISUMU strain control. 50% and 95% knockdown times were either slightly lower than those for the KISUMU strain or higher by factors of less than 1.6. The mean frequency of the East African kdr mutation was 24.7% in An. gambiae sensu strictu. Based on conventional criteria where susceptibility is defined by mortality rates >98% 24 h after exposure, no evidence for resistance was found, implying that vector control measures employing any of the insecticides tested would be unhampered by resistance. The observed frequencies of the kdr mutation do not appear to compromise the effectiveness of the insecticides. The need for continuous monitoring of the status of insecticide resistance and of the impact of any observed resistance on the efficacy of vector control programs employing insecticides is apparent.

Introduction: Emerging infectious diseases are infections that have recently appeared within a population or those whose incidence or geographic range is rapidly increasing or threatens to increase in the near future. The current study sought to re-evaluate malaria prevalence, susceptibility to ACTs, transmission patterns and the presence of malaria vectors in the Kikuyu area of the Kenyan Central highlands, a non-traditional/ low risk malaria transmission zone where there have been anecdotal reports of malaria cases The potential role of climate factors was also evaluated. The aim of the study was to generate data to inform malaria treatment policy and practice in the study area and country. Methodology: Sampling of adult mosquitoes was carried indoors by manual aspiration and using CDC light traps while mosquito larvae were sampled outdoors using larval dippers and reared to adults in the laboratory. Mosquitoes were identified by morphology and subsequently using PCR and the presence of malaria parasites in field sampled adult mosquitoes investigated using ELISA. The malaria clinical study was an open label nonrandomized clinical trial where the efficacy of one artemisinin-based antimalarial combination drug, Artemether Lumefantrine (AL) was evaluated. Two health facilities Lusigeti and Gikambura were identified for the study. Microscopy was used to identify positive cases at the health facility and nested PCR amplification targeting subunit 18s rRNA gene used to confirm positivity in the lab. P. falciparum isolates were genotyped using nested-PCR of MSP-1 (block 2) and MSP-2 (block 3) family alleles to determine the multiplicity of the infections (MOI) and characterize any subsequent infection. Antimalarial resistance gene markers Pfk13 and Pfmdr1 were analyzed Climate data for the study area was obtained from Climate Engine (http://climateengine.org) and analyzed to understand long term trends. Results: A rich repertoire of mosquito vector species was identified from the area, with the Anopheles funestus group being the predominant vector species and comprising 76.35% of all collections. Only two adult mosquitoes which were non-blood fed and negative for malaria parasites were collected. Of the 838 patients screened, 471, with a slide positivity rate of 2.1% (10) were from Lusigeti and 421, with a slide positivity rate of 7.4% (31) were from Gikambura. Parasitological analysis of microscopy outcome of the 41 cases revealed 100% (95% CI 1.96) as Adequate Clinical and Parasitological Response (ACPR). There was probable delayed parasite clearance (parasites present on Day 3) in 3(7.3%) of the cases, and no severe adverse reaction was observed. Analysis of the Pfk13 gene in the positive P. falciparum cases from the study sites revealed no SNP associated with artemisinin resistance. The pfmdr1 86Y mutation was found in 0% (0/41) of the isolates while the N86 wild allele was detected in 100%(37/37). Analysis of long term climate data showed an increase of about 1.3ºC in both the mean minimum and maximum temperatures consistent with forecasts from other sources. Conclusion: The positivity rate observed in the study site was very low but the fact that 87% of participants who tested positive did not report recent history of travel from the area and the finding of highly competent known vectors of malaria suggest a changing malaria transmission scenario requiring further investigations. That circulating parasite strains showed full sensitivity to the available treatment option indicating the absence of antimalarial drug resistance which is a positive finding.

Background Molecular diagnostic tools have been incorporated in insecticide resistance monitoring programmes to identify underlying genetic basis of resistance and develop early warning systems of vector control failure. Identifying genetic markers of insecticide resistance is crucial in enhancing the ability to mitigate potential effects of resistance. The knockdown resistance (kdr) mutation associated with resistance to DDT and pyrethroids, the acetylcholinesterase-1 (ace-1R) mutation associated with resistance to organophosphates and carbamates and 2La chromosomal inversion associated with indoor resting behaviour, were investigated in the present study. Methods Anopheles mosquitoes sampled from different sites in Kenya and collected within the context of malaria vector surveillance were analysed. Mosquitoes were collected indoors using light traps, pyrethrum spray and hand catches between August 2016 and November 2017. Mosquitoes were identified using morphological keys and Anopheles gambiae sensu lato (s.l.) mosquitoes further identified into sibling species by the polymerase chain reaction method following DNA extraction by alcohol precipitation. Anopheles gambiae and Anopheles arabiensis were analysed for the presence of the kdr and ace-1R mutations, while 2La inversion was only screened for in An. gambiae where it is polymorphic. Chi-square statistics were used to determine correlation between the 2La inversion karyotype and kdr-east mutation. Results The kdr-east mutation occurred at frequencies ranging from 0.5 to 65.6% between sites. The kdr-west mutation was only found in Migori at a total frequency of 5.3% (n = 124). No kdr mutants were detected in Tana River. The ace-1R mutation was absent in all populations. The 2La chromosomal inversion screened in An. gambiae occurred at frequencies of 87% (n = 30), 80% (n = 10) and 52% (n = 50) in Baringo, Tana River and Migori, respectively. A significant association between the 2La chromosomal inversion and the kdr-east mutation was found. Conclusion The significant association between the 2La inversion karyotype and kdr-east mutation suggests that pyrethroid resistant An. gambiae continue to rest indoors regardless of the presence of treated bed nets and residual sprays, a persistence further substantiated by studies documenting continued mosquito abundance indoors. Behavioural resistance by which Anopheles vectors prefer not to rest indoors may, therefore, not be a factor of concern in this study’s malaria vector populations.

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