Chlorfenapyr metabolism by mosquito P450s associated with pyrethroid resistance identifies potential activation markers.

Chlorfenapyr is a pro-insecticide increasingly used in combination with pyrethroids such as a-cypermethrin or deltamethrin in insecticide treated bednets (ITNs) to control malaria transmitted by pyrethroid-resistant mosquito populations. Chlorfenapyr requires P450 activation to produce tralopyril and other bioactive metabolites. Pyrethroid resistance is often associated with elevated levels of chemoprotective P450s with broad substrate specificity, which could influence chlorfenapyr activity. Here, we have investigated chlorfenapyr metabolism by a panel of eight P450s commonly associated with pyrethroid resistance in An. gambiae and Ae. aegypti, the major vectors of malaria and arboviruses. Chlorfenapyr was activated to tralopyril by An. gambiae CYP6P3, CYP9J5, CYP9K1 and Ae. aegypti, CYP9J32. The Kcat/KM value of 0.66 µM-1 min-1 for CYP9K1 was, 6.7 fold higher than CYP6P3 and CYP9J32 (both 0.1 µM-1 min-1) and 22-fold higher than CYP9J5 (0.03 µM-1 min-1). Further investigation of the effect of -cypermethrin equivalent to the ratios used with chlorfenapyr in bed nets (~ 1:2 molar ratio) resulted in a reduction in chlorfenapyr metabolism by CYP6P3 and CYP6K1 of 76.8% and 56.8% respectively. This research provides valuable insights into the metabolism of chlorfenapyr by mosquito P450s and highlights the need for continued investigation into effective vector control strategies.

Resilience of transfluthrin to oxidative attack by duplicated CYP6P9 variants known to confer pyrethroid resistance in the major malaria mosquito Anopheles funestus.

Resistance to common pyrethroids, such as deltamethrin and permethrin is widespread in the malaria mosquito Anopheles funestus and mainly conferred by upregulated cytochrome P450 monooxygenases (P450s). In the pyrethroid resistant laboratory strain An. funestus FUMOZ-R the duplicated genes CYP6P9a and CYP6P9b are highly upregulated and have been shown to metabolize various pyrethroids, including deltamethrin and permethrin. Here, we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus using a baculovirus expression system and evaluated the interaction of the multifluorinated benzyl pyrethroid transfluthrin with these enzymes by different approaches. First, by Michaelis-Menten kinetics in a fluorescent probe assay with the model substrate 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC), we showed the inhibition of BOMFC metabolism by increasing concentrations of transfluthrin. Second, we tested the metabolic capacity of recombinantly expressed CYP6P9 variants to degrade transfluthrin utilizing UPLC-MS/MS analysis and detected low depletion rates, explaining the virtual lack of resistance of strain FUMOZ-R to transfluthrin observed in previous studies. However, as both approaches suggested an interaction of CYP6P9 variants with transfluthrin, we analyzed the oxidative metabolic fate and failed to detect hydroxylated transfluthrin, but low amounts of an M-2 transfluthrin metabolite. Based on the detected metabolite we hypothesize oxidative attack of the gem-dimethyl substituted cyclopropyl moiety, resulting in the formation of an allyl cation upon ring opening. In conclusion, these findings support the resilience of transfluthrin to P450-mediated pyrethroid resistance, and thus, reinforces its employment as an important resistance-breaking pyrethroid in resistance management strategies to control the major malaria vector An. funestus.

Tandem duplication of a genomic region encoding glutathione S-transferase epsilon-2 and -4 genes in DDT-resistant Anopheles stephensi strain from India.

The glutathione S-transferases (GST) genes are a multigene family of enzymes involved in the metabolism of endogenous and xenobiotic compounds by catalysing the conjugation of the reduced form of glutathione to the substrate. The epsilon class of GST (GSTe), unique to arthropods, is known to be involved in the detoxification process of several classes of insecticides, and GSTe2 in particular is known to have DDT dehydrochlorinase activity. This communication reports a tandem duplication of a genomic region encoding GSTe2 and GSTe4 genes in a laboratory-colonized DDT-resistant Anopheles stephensi. We identified duplication breakpoints and the organization of gene duplication through Sanger sequencing performed on long-PCR products. Manual annotation of sequences revealed a tandemly-arrayed duplication of a 3.62 kb segment of GST epsilon gene clusters comprised of five genes: a partial GSTe1, GSTe2, GSTe2-pseudogene, GSTe4 and partial GSTe5, interconnected by a conserved 2.42 kb DNA insert segment major part of which is homologous to a genomic region located on a different chromosome. The tandemly duplicated array contained a total of two GSTe2 and three GSTe4 functional paralog genes. Read-depth coverage and split-read analysis of Illumina-based whole-genome sequence reads confirmed the presence of duplication in the corresponding region of the genome. The increased gene dose in mosquitoes as a result of the GSTe gene-duplication may be an adaptive process to increase levels of detoxifying enzymes to counter insecticide pressure.

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

Studies of insecticide resistance provide insights into the capacity of populations to show rapid evolutionary responses to contemporary selection. Malaria control remains heavily dependent on pyrethroid insecticides, primarily in long lasting insecticidal nets (LLINs). Resistance in the major malaria vectors has increased in concert with the expansion of LLIN distributions. Identifying genetic mechanisms underlying high-level resistance is crucial for the development and deployment of resistance-breaking tools. Using the Anopheles gambiae 1000 genomes (Ag1000g) data we identified a very recent selective sweep in mosquitoes from Uganda which localized to a cluster of cytochrome P450 genes. Further interrogation revealed a haplotype involving a trio of mutations, a nonsynonymous point mutation in Cyp6p4 (I236M), an upstream insertion of a partial Zanzibar-like transposable element (TE) and a duplication of the Cyp6aa1 gene. The mutations appear to have originated recently in An. gambiae from the Kenya-Uganda border, with stepwise replacement of the double-mutant (Zanzibar-like TE and Cyp6p4-236 M) with the triple-mutant haplotype (including Cyp6aa1 duplication), which has spread into the Democratic Republic of Congo and Tanzania. The triple-mutant haplotype is strongly associated with increased expression of genes able to metabolize pyrethroids and is strongly predictive of resistance to pyrethroids most notably deltamethrin. Importantly, there was increased mortality in mosquitoes carrying the triple-mutation when exposed to nets cotreated with the synergist piperonyl butoxide (PBO). Frequencies of the triple-mutant haplotype remain spatially variable within countries, suggesting an effective marker system to guide deployment decisions for limited supplies of PBO-pyrethroid cotreated LLINs across African countries.

Sequential phase I metabolism of pyrethroids by duplicated CYP6P9 variants results in the loss of the terminal benzene moiety and determines resistance in the malaria mosquito Anopheles funestus.

Pyrethroid resistance in Anopheles funestus is threatening the eradication of malaria. One of the major drivers of pyrethroid resistance in An. funestus are cytochrome P450 monooxygenases CYP6P9a and CYP6P9b, which are found upregulated in resistant An. funestus populations from Sub-Saharan Africa and are known to metabolise pyrethroids. Here, we have functionally expressed CYP6P9a and CYP6P9b variants and investigated their interactions with azole-fungicides and pyrethroids. Some azole fungicides such as prochloraz inhibited CYP6P9a and CYP6P9b at nanomolar concentrations, whereas pyrethroids were weak inhibitors (>100 µM). Amino acid sequence comparisons suggested that a valine to isoleucine substitution at position 310 in the active site cavity of CYP6P9a and CYP6P9b, respectively, might affect substrate binding and metabolism. We therefore swapped the residues by site directed mutagenesis to produce CYP6P9aI310V and CYP6P9bV310I. CYP6P9bV310I produced stronger metabolic activity towards coumarin substrates and pyrethroids, particularly permethrin. The V310I mutation was previously also detected in a pyrethroid resistant field population of An. funestus in Benin. Additionally, we found the first metabolite of permethrin and deltamethrin after hydroxylation, 4'OH permethrin and 4'OH deltamethrin, were also suitable substrates for CYP6P9-variants, and were depleted by both enzymes to a higher extent than as their respective parent compounds (approximately 20% more active). Further, we found that both metabolites were toxic against An. funestus FANG (pyrethroid susceptible) but not towards FUMOZ-R (pyrethroid resistant) mosquitoes, the latter suggesting detoxification by overexpressed CYP6P9a and CYP6P9b. We confirmed by mass-spectrometric analysis that CYP6P9a and CYP6P9b are capable of cleaving phenoxybenzyl-ethers in type I pyrethroid permethrin and type II pyrethroid deltamethrin and that both enzymes preferentially metabolise trans-permethrin. This provides new insight into the metabolism of pyrethroids and a greater understanding of the molecular mechanisms of pyrethroid resistance in An. funestus.

A high-throughput HPLC method for simultaneous quantification of pyrethroid and pyriproxyfen in long-lasting insecticide-treated nets.

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.

Biochemical profiling of functionally expressed CYP6P9 variants of the malaria vector Anopheles funestus with special reference to cytochrome b5 and its role in pyrethroid and coumarin substrate metabolism.

Cytochrome P450 monooxygenases (P450s) are well studied enzymes catalyzing the oxidative metabolism of xenobiotics in insects including mosquitoes. Their duplication and upregulation in agricultural and public health pests such as anopheline mosquitoes often leads to an enhanced metabolism of insecticides which confers resistance. In the laboratory strain Anopheles funestus FUMOZ-R the duplicated P450s CYP6P9a and CYP6P9b are highly upregulated and proven to confer pyrethroid resistance. Microsomal P450 activity is regulated by NADPH cytochrome P450 oxidoreductase (CPR) required for electron transfer, whereas the modulatory role of cytochrome b5 (CYB5) on insect P450 activity is less clear. In previous studies CYP6P9a and CYP6P9b were recombinantly expressed in tandem with An. gambiae CPR using E. coli-expression systems and CYB5 added to the reaction mix to enhance activity. However, the precise role of CYB5 on substrate turn-over when combined with CYP6P9a and CYP6P9b remains poorly investigated, thus one objective of our study was to address this knowledge gap. In contrast to the CYP6P9 variants, the expression levels of both CYB5 and CPR were not upregulated in the pyrethroid resistant FUMOZ-R strain when compared to the susceptible FANG strain, suggesting no immediate regulatory role of these genes in pyrethroid resistance in FUMOZ-R. Here, for the first time we recombinantly expressed CYP6P9a and CYP6P9b from An. funestus in a baculovirus expression system using High-5 insect cells. Co-expression of each enzyme with CPR from either An. gambiae or An. funestus did not reveal noteworthy differences in catalytic capacity. Whereas the co-expression of An. funestus CYB5 - tested at different multiplicity of infection (MOI) ratios - resulted in a significantly higher metabolization of coumarin substrates as measured by fluorescence assays. This was confirmed by Michaelis-Menten kinetics using the most active substrate, 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC). We observed a similar increase in coumarin substrate turnover by adding human CYB5 to the reaction mix. Finally, we compared by UPLC-MS/MS analysis the depletion rate of deltamethrin and the formation of 4'OH-deltamethrin by recombinantly expressed CYP6P9a and CYP6P9b with and without CYB5 and detected no difference in the extent of deltamethrin metabolism. Our results suggest that co-expression (or addition) of CYB5 with CYP6P9 variants, recombinantly expressed in insect cells, can significantly enhance their metabolic capacity to oxidize coumarins, but not deltamethrin.

Review and Meta-Analysis of the Evidence for Choosing between Specific Pyrethroids for Programmatic Purposes.

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.

Indoor residual spraying practices against Triatoma infestans in the Bolivian Chaco: contributing factors to suboptimal insecticide delivery to treated households.

BACKGROUND: 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. AIMS: This study evaluated routine IRS practices and insecticide quality control in a typical endemic community in the Bolivian Chaco. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Improving houses in the Bolivian Chaco increases effectiveness of residual insecticide spraying against infestation with Triatoma infestans, vector of Chagas disease.

OBJECTIVE: Failure to control domestic Triatoma infestans in the Chaco is attributed to vulnerable adobe construction, which provides vector refuges and diminishes insecticide contact. We conducted a pilot to test the impact of housing improvement plus indoor residual spraying (IRS) on house infestation and vector abundance in a rural community in the Bolivian Chaco. METHODS: The intervention included three arms: housing improvement + IRS [HI], assisted IRS [AS] in which the team helped to clear the house pre-IRS and routine IRS [RS]. HI used locally available materials, traditional construction techniques and community participation. Vector parameters were assessed by Timed Manual Capture for 2 person-hours per house at baseline and medians of 114, 173, 314, 389 and 445 days post-IRS-1. A second IRS round was applied at a median of 314 days post-IRS-1. RESULTS: Post-intervention infestation indices and abundance fell in all three arms. The mean odds of infestation was 0.29 (95% CL 0.124, 0.684) in the HI relative to the RS arm. No difference was observed between AS and RS. Vector abundance was reduced by a mean 44% (24.8, 58.0) in HI compared to RS, with no difference between AS and RS. Median delivered insecticide concentrations per house were lower than the target of 50 mg/m2 in >90% of houses in all arms. CONCLUSION: Housing improvement using local materials and community participation is a promising strategy to improve IRS effectiveness in the Bolivian Chaco. A larger trial is needed to quantify the impact on reinfestation over time.

High pyrethroid/DDT resistance in major malaria vector Anopheles coluzzii from Niger-Delta of Nigeria is probably driven by metabolic resistance mechanisms.

Entomological surveillance of local malaria vector populations is an important component of vector control and resistance management. In this study, the resistance profile and its possible mechanisms was characterised in a field population of the major malaria vector Anopheles coluzzii from Port Harcourt, the capital of Rivers state, in the Niger-Delta Region of Nigeria. Larvae collected in Port-Harcourt, were reared to adulthood and used for WHO bioassays. The population exhibited high resistance to permethrin, deltamethrin and DDT with mortalities of 6.7% ± 2.4, 37.5% ± 3.2 and 6.3% ± 4.1, respectively, but were fully susceptible to bendiocarb and malathion. Synergist bioassays with piperonylbutoxide (PBO) partially recovered susceptibility, with mortalities increasing to 53% ± 4, indicating probable role of CYP450s in permethrin resistance (χ2 = 29.48, P < 0.0001). Transcriptional profiling revealed five major resistance-associated genes overexpressed in the field samples compared to the fully susceptible laboratory colony, Ngoussou. Highest fold change (FC) was observed with GSTe2 (FC = 3.3 in permethrin exposed and 6.2 in unexposed) and CYP6Z3 (FC = 1.4 in exposed and 4.6 in unexposed). TaqMan genotyping of 32 F0 females detected the 1014F and 1575Y knockdown resistance (kdr) mutations with frequencies of 0.84 and 0.1, respectively, while 1014S mutation was not detected. Sequencing of a fragment of the voltage-gated sodium channel, spanning exon 20 from 13 deltamethrin-resistant and 9 susceptible females revealed only 2 distinct haplotypes with a low haplotype diversity of 0.33. The findings of high pyrethroid resistance but with a significant degree of recovery after PBO synergist assay suggests the need to move to PBO-based nets. This could be complemented with carbamate- or organophosphate-based indoor residual spraying in this area.

High concentrations of membrane-fed ivermectin are required for substantial lethal and sublethal impacts on Aedes aegypti.

BACKGROUND: With widespread insecticide resistance in mosquito vectors, there is a pressing need to evaluate alternatives with different modes of action. Blood containing the antihelminthic drug ivermectin has been shown to have lethal and sub-lethal effects on mosquitoes. Almost all work to date has been on Anopheles spp., but impacts on other anthropophagic vectors could provide new options for their control, or additional value to anti-malarial ivermectin programmes. METHODS: Using dose-response assays, we evaluated the effects of ivermectin delivered by membrane feeding on daily mortality (up to 14 days post-blood feed) and fecundity of an Indian strain of Aedes aegypti. RESULTS: The 7-day lethal concentration of ivermectin required to kill 50% of adult mosquitoes was calculated to be 178.6 ng/ml (95% confidence intervals 142.3-218.4) for Ae. aegypti, which is much higher than that recorded for Anopheles spp. in any previous study. In addition, significant effects on fecundity and egg hatch rates were only recorded at high ivermectin concentrations (≥ 250 ng/ul). CONCLUSION: Our results suggest that levels of ivermectin present in human blood at current dosing regimes in mass drug administration campaigns, or even those in a recent higher-dose anti-malaria trial, are unlikely to have a substantial impact on Ae. aegypti. Moreover, owing to the strong anthropophagy of Ae. aegypti, delivery of higher levels of ivermectin in livestock blood is also unlikely to be an effective option for its control. However, other potential toxic impacts of ivermectin metabolites, accumulation in tissues, sublethal effects on behaviour, or antiviral action might increase the efficacy of ivermectin against Ae. aegypti and the arboviral diseases it transmits, and require further investigation.

Towards understanding transfluthrin efficacy in a pyrethroid-resistant strain of the malaria vector Anopheles funestus with special reference to cytochrome P450-mediated detoxification.

Malaria vector control interventions rely heavily on the application of insecticides against anopheline mosquitoes, in particular the fast-acting pyrethroids that target insect voltage-gated sodium channels (VGSC). Frequent applications of pyrethroids have resulted in resistance development in the major malaria vectors including Anopheles funestus, where resistance is primarily metabolic and driven by the overexpression of microsomal cytochrome P450 monooxygenases (P450s). Here we examined the pattern of cross-resistance of the pyrethroid-resistant An. funestus strain FUMOZ-R towards transfluthrin and multi-halogenated benzyl derivatives, permethrin, cypermethrin and deltamethrin in comparison to the susceptible reference strain FANG. Transfluthrin and two multi-fluorinated derivatives exhibited micromolar potency - comparable to permethrin - to functionally expressed dipteran VGSC in a cell-based cation influx assay. The activity of transfluthrin and its derivatives on VGSC was strongly correlated with their contact efficacy against strain FUMOZ-R, although no such correlation was obtained for the other pyrethroids due to their rapid detoxification by the resistant strain. The low resistance levels for transfluthrin and derivatives in strain FUMOZ-R were only weakly synergized by known P450 inhibitors such as piperonyl butoxide (PBO), triflumizole and 1-aminobenzotriazole (1-ABT). In contrast, deltamethrin toxicity in FUMOZ-R was synergized > 100-fold by all three P450 inhibitors. The biochemical profiling of a range of fluorescent resorufin and coumarin compounds against FANG and FUMOZ-R microsomes identified 7-benzyloxymethoxy-4-trifluoromethylcoumarin (BOMFC) as a highly sensitive probe substrate for P450 activity. BOMFC was used to develop a fluorescence-based high-throughput screening assay to measure the P450 inhibitory action of potential synergists. Azole fungicides prochloraz and triflumizole were identified as extremely potent nanomolar inhibitors of microsomal P450s, strongly synergizing deltamethrin toxicity in An. funestus. Overall, the present study contributed to the understanding of transfluthrin efficacy at the molecular and organismal level and identified azole compounds with potential to synergize pyrethroid efficacy in malaria vectors.

New insecticide screening platforms indicate that Mitochondrial Complex I inhibitors are susceptible to cross-resistance by mosquito P450s that metabolise pyrethroids.

Fenazaquin, pyridaben, tolfenpyrad and fenpyroximate are Complex I inhibitors offering a new mode of action for insecticidal malaria vector control. However, extended exposure to pyrethroid based products such as long-lasting insecticidal nets (LLINs) has created mosquito populations that are largely pyrethroid-resistant, often with elevated levels of P450s that can metabolise and neutralise diverse substrates. To assess cross-resistance liabilities of the Complex I inhibitors, we profiled their susceptibility to metabolism by P450s associated with pyrethroid resistance in Anopheles gambiae (CYPs 6M2, 6P3, 6P4, 6P5, 9J5, 9K1, 6Z2) and An. funestus (CYP6P9a). All compounds were highly susceptible. Transgenic An. gambiae overexpressing CYP6M2 or CYP6P3 showed reduced mortality when exposed to fenpyroximate and tolfenpyrad. Mortality from fenpyroximate was also reduced in pyrethroid-resistant strains of An. gambiae (VK7 2014 and Tiassalé 13) and An. funestus (FUMOZ-R). P450 inhibitor piperonyl butoxide (PBO) significantly enhanced the efficacy of fenpyroximate and tolfenpyrad, fully restoring mortality in fenpyroximate-exposed FUMOZ-R. Overall, results suggest that in vivo and in vitro assays are a useful guide in the development of new vector control products, and that the Complex I inhibitors tested are susceptible to metabolic cross-resistance and may lack efficacy in controlling pyrethroid resistant mosquitoes.

Improving the performance of spray operators through monitoring and evaluation of insecticide concentrations of pirimiphos-methyl during indoor residual spraying for malaria control on Bioko Island.

BACKGROUND: Quality control of indoor residual spraying (IRS) is necessary to ensure that spray operators (SOs) deposit the correct concentration of insecticide on sprayed structures, while also confirming that spray records are not being falsified. METHODS: Using high-performance liquid chromatography (HPLC), this study conducted quality control of the organophosphate insecticide pirimiphos-methyl (Actellic 300CS), during the 2018 IRS round on Bioko Island, Equatorial Guinea. Approximately 60 SOs sprayed a total of 67,721 structures in 16,653 houses during the round. Houses that were reportedly sprayed were randomly selected for quality control testing. The SOs were monitored twice in 2018, an initial screening in March followed by sharing of results with the IRS management team and identification of SOs to be re-trained, and a second screening in June to monitor the effectiveness of training. Insecticide samples were adhesive-lifted from wooden and cement structures and analysed using HPLC. RESULTS: The study suggests that with adequate quality control measures and refresher training, suboptimal spraying was curtailed, with a significant increased concentration delivered to the bedroom (difference = 0.36, P < 0.001) and wooden surfaces (difference 0.41, P = 0.001). Additionally, an increase in effective coverage by SOs was observed, improving from 80.7% in March to 94.7% in June after re-training (McNemar's test; P = 0.03). CONCLUSIONS: The ability to randomly select, locate, and test houses reportedly sprayed within a week via HPLC has led to improvements in the performance of SOs on Bioko Island, enabling the project to better evaluate its own performance.

Cross-resistance profiles of malaria mosquito P450s associated with pyrethroid resistance against WHO insecticides.

Extensive use of pyrethroids for malaria control in Africa has led to widespread pyrethroid resistance in the two major African vectors of malaria An. gambiae and An. funestus. This is often associated with constitutively elevated levels of cytochrome P450s involved with pyrethroid metabolism and detoxification. P450s have the capacity to metabolise diverse substrates, which raises concerns about their potential to cause cross-resistance. A bank of seven recombinant P450s from An. gambiae (CYPs 6M2, 6P2, 6P3, 6P4, 6P5, 9J5) and An. funestus (CYP6P9a) commonly associated with pyrethroid resistance were screened against twelve insecticides representing the five major classes of insecticides recommended by WHO for malaria control; permethrin, etofenprox and bifenthrin (type I pyrethroids), deltamethrin, lambda cyhalothrin and cypermethrin (type II pyrethroids), DDT (organochlorine), bendiocarb (carbamate), malathion, pirimiphos methyl and fenitrothion (organophosphates) and pyriproxyfen (juvenile hormone analogue). DDT was not metabolised by the P450 panel, while bendiocarb was only metabolised by CYP6P3. Pyrethroids and pyriproxyfen were largely susceptible to metabolism by the P450 panel, as were organophosphates, which are activated by P450s. Primiphos-methyl is increasingly used for malaria control. Examination of the pirimiphos-methyl metabolites generated by CYP6P3 revealed both the active pirimiphos-methyl-oxon form and the inactive oxidative cleavage product 2-diethylamino-6-hydroxy-4-methylpyrimidine. The inhibition profile of CYPs 6M2, 6P2, 6P3, 6P9a and 9J5 was also examined using diethoxyfluorescein (DEF) as the probe substrate. Bendiocarb was the weakest inhibitor with IC50 > 100 µM across the P450 panel, while CYP6M2 showed strongest inhibition by malathion (IC50 0.7 µM). The results suggest that P450s present at elevated levels in two major Anopheline vectors of malaria in Africa have the capacity to metabolise a diverse range of pyrethroid and organophosphate insecticides as well as pyriproxyfen that could impact vector control.

Antimalarial activity of primaquine operates via a two-step biochemical relay.

Primaquine (PQ) is an essential antimalarial drug but despite being developed over 70 years ago, its mode of action is unclear. Here, we demonstrate that hydroxylated-PQ metabolites (OH-PQm) are responsible for efficacy against liver and sexual transmission stages of Plasmodium falciparum. The antimalarial activity of PQ against liver stages depends on host CYP2D6 status, whilst OH-PQm display direct, CYP2D6-independent, activity. PQ requires hepatic metabolism to exert activity against gametocyte stages. OH-PQm exert modest antimalarial efficacy against parasite gametocytes; however, potency is enhanced ca.1000 fold in the presence of cytochrome P450 NADPH:oxidoreductase (CPR) from the liver and bone marrow. Enhancement of OH-PQm efficacy is due to the direct reduction of quinoneimine metabolites by CPR with the concomitant and excessive generation of H2O2, leading to parasite killing. This detailed understanding of the mechanism paves the way to rationally re-designed 8-aminoquinolines with improved pharmacological profiles.

Development of a rapid field-applicable molecular diagnostic for knockdown resistance (kdr) markers in An. gambiae.

BACKGROUND: The spread of insecticide resistance (IR) is a major threat to vector control programmes for mosquito-borne diseases. Early detection of IR using diagnostic markers could help inform these programmes, especially in remote locations where gathering reliable bioassay data is challenging. Most current molecular tests for genetic IR markers are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics. METHODS: A single-cartridge test was designed to detect key IR mutations in the major African vector of malaria, Anopheles gambiae. Developed on the portable, rapid, point-of-care compatible PCR platform - Genedrive® (genedrive® plc), the test comprises two assays which target single nucleotide polymorphisms (SNPs) in the voltage gated sodium channel (VGSC) gene that exert interactive effects on knockdown resistance (kdr): L1014F, L1014S and N1575Y. RESULTS: Distinct melt peaks were observed for each allele at each locus. Preliminary validation of these assays using a test panel of 70 An. gambiae samples showed complete agreement of our assays with the widely-used TaqMan assays, achieving a sensitivity and specificity of 100%. CONCLUSION: Here we show the development of an insecticide resistance detection assay for use on the Genedrive® platform that has the potential to be the first field-applicable diagnostic for kdr.