Isoliensinine from Cissampelos pariera rhizomes exhibits potential gametocytocidal and anti-malarial activities against Plasmodium falciparum clinical isolates.

BACKGROUND: The unmet demand for effective malaria transmission-blocking agents targeting the transmissible stages of Plasmodium necessitates intensive discovery efforts. In this study, a bioactive bisbenzylisoquinoline (BBIQ), isoliensinine, from Cissampelos pariera (Menispermaceae) rhizomes was identified and characterized for its anti-malarial activity. METHODS: Malaria SYBR Green I fluorescence assay was performed to evaluate the in vitro antimalarial activity against D6, Dd2, and F32-ART5 clones, and immediate ex vivo (IEV) susceptibility for 10 freshly collected P. falciparum isolates. To determine the speed- and stage-of-action of isoliensinine, an IC50 speed assay and morphological analyses were performed using synchronized Dd2 asexuals. Gametocytocidal activity against two culture-adapted gametocyte-producing clinical isolates was determined using microscopy readouts, with possible molecular targets and their binding affinities deduced in silico. RESULTS: Isoliensinine displayed a potent in vitro gametocytocidal activity at mean IC50gam values ranging between 0.41 and 0.69 µM for Plasmodium falciparum clinical isolates. The BBIQ compound also inhibited asexual replication at mean IC50Asexual of 2.17 µM, 2.22 µM, and 2.39 µM for D6, Dd2 and F32-ART5 respectively, targeting the late-trophozoite to schizont transition. Further characterization demonstrated a considerable immediate ex vivo potency against human clinical isolates at a geometric mean IC50IEV = 1.433 µM (95% CI 0.917-2.242). In silico analyses postulated a probable anti-malarial mechanism of action by high binding affinities for four mitotic division protein kinases; Pfnek1, Pfmap2, Pfclk1, and Pfclk4. Additionally, isoliensinine was predicted to possess an optimal pharmacokinetics profile and drug-likeness properties. CONCLUSION: These findings highlight considerable grounds for further exploration of isoliensinine as an amenable scaffold for malaria transmission-blocking chemistry and target validation.

Neurotoxic Zanthoxylum chalybeum root constituents invoke mosquito larval growth retardation through ecdysteroidogenic CYP450s transcriptional perturbations.

Intracellular effects exerted by phytochemicals eliciting insect growth-retarding responses during vector control intervention remain largely underexplored. We studied the effects of Zanthoxylum chalybeum Engl. (Rutaceae) (ZCE) root derivatives against malaria (Anopheles gambiae) and arbovirus vector (Aedes aegypti) larvae to decipher possible molecular targets. We report dose-dependent biphasic effects on larval response, with transient exposure to ZCE and its bioactive fraction (ZCFr.5) inhibiting acetylcholinesterase (AChE) activity, inducing larval lethality and growth retardation at sublethal doses. Half-maximal lethal concentrations (LC50) for ZCE against An. gambiae and Ae. aegypti larvae after 24-h exposure were 9.00 ppm and 12.26 ppm, respectively. The active fraction ZCFr.5 exerted LC50 of 1.58 ppm and 3.21 ppm for An. gambiae and Ae. aegypti larvae, respectively. Inhibition of AChE was potentially linked to larval toxicity afforded by 2-tridecanone, palmitic acid (hexadecanoic acid), linoleic acid ((Z,Z)-9,12-octadecadienoic acid), sesamin, ß-caryophyllene among other compounds identified in the bioactive fraction. In addition, the phenotypic larval retardation induced by ZCE root constituents was exerted through transcriptional modulation of ecdysteroidogenic CYP450 genes. Collectively, these findings provide an explorative avenue for developing potential mosquito control agents from Z. chalybeum root constituents.

Community knowledge and perceptions on malaria prevention and house screening in Nyabondo, Western Kenya.

BACKGROUND: Screening of houses to prevent mosquito entry is increasingly being recommended as an effective and practical method against malaria transmission through reduced human-mosquito contact. The objective of the study was to assess community knowledge and perceptions on malaria prevention and house screening in a malaria endemic area of Western Kenya. METHODS: A cross-sectional household survey was conducted in 2017 in Nyabondo area of western Kenya. A total of 80 households were randomly selected to participate in the study within 16 villages. Structured questionnaires, focus group discussions and key informant interviews were used to collect data. RESULTS: A total of 80 respondents participated in the survey and more than half (53.8%) reported to have attained primary education. About 91% of the respondents had previously seen or heard malaria messages and this was associated with the respondents level of education (χ2 = 10.163; df 4; P = 0.038, 95% CI). However, other variables like gender, marital status, religion and occupation were not significantly associated with knowledge in malaria. Insecticide treated mosquito nets was by far the most reported known (97.4%) and applied (97.6%) personal protective while only 15.6% respondents were aware of house screening. The major reason given for screening doors, windows and eaves was to prevent entry of mosquito and other insects (> 85%). Lack of awareness was the major reason given for not screening houses. Grey colour was the most preferred choice for screen material (48%), and the main reason given was that grey matched the colour of the walls (21%) and did not 'gather' dust quickly. CONCLUSION: House screening was not a common intervention for self-protection against malaria vectors in the study area. There is need to advocate and promote house screening to increase community knowledge on this as an additional integrated vector management strategy for malaria control.

Spatial panorama of malaria prevalence in Africa under climate change and interventions scenarios.

BACKGROUND: Malaria is highly sensitive to climatic variables and is strongly influenced by the presence of vectors in a region that further contribute to parasite development and sustained disease transmission. Mathematical analysis of malaria transmission through the use and application of the value of the basic reproduction number (R0) threshold is an important and useful tool for the understanding of disease patterns. METHODS: Temperature dependence aspect of R0 obtained from dynamical mathematical network model was used to derive the spatial distribution maps for malaria transmission under different climatic and intervention scenarios. Model validation was conducted using MARA map and the Annual Plasmodium falciparum Entomological Inoculation Rates for Africa. RESULTS: The inclusion of the coupling between patches in dynamical model seems to have no effects on the estimate of the optimal temperature (about 25 °C) for malaria transmission. In patches environment, we were able to establish a threshold value (about α = 5) representing the ratio between the migration rates from one patch to another that has no effect on the magnitude of R0. Such findings allow us to limit the production of the spatial distribution map of R0 to a single patch model. Future projections using temperature changes indicated a shift in malaria transmission areas towards the southern and northern areas of Africa and the application of the interventions scenario yielded a considerable reduction in transmission within malaria endemic areas of the continent. CONCLUSIONS: The approach employed here is a sole study that defined the limits of contemporary malaria transmission, using R0 derived from a dynamical mathematical model. It has offered a unique prospect for measuring the impacts of interventions through simple manipulation of model parameters. Projections at scale provide options to visualize and query the results, when linked to the human population could potentially deliver adequate highlight on the number of individuals at risk of malaria infection across Africa. The findings provide a reasonable basis for understanding the fundamental effects of malaria control and could contribute towards disease elimination, which is considered as a challenge especially in the context of climate change.

Select ß- and γ-branched 1-alkylpyrazol-4-yl methylcarbamates exhibit high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

The widespread emergence of pyrethroid-resistant Anopheles gambiae has intensified the need to find new contact mosquitocides for indoor residual spraying and insecticide treated nets. With the goal of developing new species-selective and resistance-breaking acetylcholinesterase (AChE)-inhibiting mosquitocides, in this report we revisit the effects of carbamate substitution on aryl carbamates, and variation of the 1-alkyl group on pyrazol-4-yl methylcarbamates. Compared to aryl methylcarbamates, aryl dimethylcarbamates were found to have lower selectivity for An. gambiae AChE (AgAChE) over human AChE (hAChE), but improved tarsal contact toxicity to G3 strain An. gambiae. Molecular modeling studies suggest the lower species-selectivity of the aryl dimethylcarbamates can be attributed to a less flexible acyl pocket in AgAChE relative to hAChE. The improved tarsal contact toxicity of the aryl dimethylcarbamates relative to the corresponding methylcarbamates is attributed to a range of complementary phenomena. With respect to the pyrazol-4-yl methylcarbamates, the previously observed low An. gambiae-selectivity of compounds bearing α-branched 1-alkyl groups was improved by employing ß- and γ-branched 1-alkyl groups. Compounds 22a (cyclopentylmethyl), 21a (cyclobutylmethyl), and 26a (3-methylbutyl) offer 250-fold, 120-fold, and 96-fold selectivity, respectively, for inhibition of AgAChE vs. hAChE. Molecular modeling studies suggests the high species-selectivity of these compounds can be attributed to the greater mobility of the W84 side chain in the choline-binding site of AgAChE, compared to that of W86 in hAChE. Compound 26a has reasonable contact toxicity to G3 strain An. gambiae (LC50 = 269 µg/mL) and low cross-resistance to Akron strain (LC50 = 948 µg/mL), which bears the G119S resistance mutation.

Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya.

BACKGROUND: Small islands serve as potential malaria reservoirs through which new infections might come to the mainland and may be important targets in malaria elimination efforts. This study investigated malaria vector species diversity, blood-meal hosts, Plasmodium infection rates, and long-lasting insecticidal net (LLIN) coverage on Mageta, Magare and Ngodhe Islands of Lake Victoria in western Kenya, a region where extensive vector control is implemented on the mainland. RESULTS: From trapping for six consecutive nights per month (November 2012 to March 2015) using CDC light traps, pyrethrum spray catches and backpack aspiration, 1868 Anopheles mosquitoes were collected. Based on their cytochrome oxidase I (COI) and intergenic spacer region PCR and sequencing, Anopheles gambiae s.l. (68.52%), Anopheles coustani (19.81%) and Anopheles funestus s.l. (11.67%) mosquitoes were differentiated. The mean abundance of Anopheles mosquitoes per building per trap was significantly higher (p < 0.001) in Mageta than in Magare and Ngodhe. Mageta was also the most populated island (n = 6487) with low LLIN coverage of 62.35% compared to Ngodhe (n = 484; 88.31%) and Magare (n = 250; 98.59%). Overall, 416 (22.27%) engorged Anopheles mosquitoes were analysed, of which 41 tested positive for Plasmodium falciparum infection by high-resolution melting (HRM) analysis of 18S rRNA and cytochrome b PCR products. Plasmodium falciparum infection rates were 10.00, 11.76, 0, and 18.75% among blood-fed An. gambiae s.s. (n = 320), Anopheles arabiensis (n = 51), An. funestus s.s. (n = 29), and An. coustani (n = 16), respectively. Based on HRM analysis of vertebrate cytochrome b, 16S rRNA and COI PCR products, humans (72.36%) were the prominent blood-meal hosts of malaria vectors, but 20.91% of blood-meals were from non-human vertebrate hosts. CONCLUSIONS: These findings demonstrate high Plasmodium infection rates among the primary malaria vectors An. gambiae s.s. and An. arabiensis, as well as in An. coustani for the first time in the region, and that non-human blood-meal sources play an important role in their ecology. Further, the higher Anopheles mosquito abundances on the only low LLIN coverage island of Mageta suggests that high LLIN coverage has been effective in reducing malaria vector populations on Magare and Ngodhe Islands.

Erratum to: Composition of Anopheles mosquitoes, their blood-meal hosts, and Plasmodium falciparum infection rates in three islands with disparate bed net coverage in Lake Victoria, Kenya.

After publication of the article [1], it has been brought to our attention that a funding acknowledgement has been omitted from the original article. The authors would like to include the following, "The study was undertaken as part of the Target Malaria consortium, which receives core funding from the Bill & Melinda Gates Foundation & from the Open Philanthropy Project Fund, an advised fund of Silicon Valley Community Foundation."

Eave tubes for malaria control in Africa: prototyping and evaluation against Anopheles gambiae s.s. and Anopheles arabiensis under semi-field conditions in western Kenya.

BACKGROUND: Whilst significant progress has been made in the fight against malaria, vector control continues to rely on just two insecticidal methods, i.e., indoor residual spraying and insecticidal bed nets. House improvement shows great potential to complement these methods and may further reduce indoor mosquito biting and disease transmission. Open eaves serve as important mosquito house entry points and provide a suitable location for intercepting host-seeking anophelines. This study describes semi-field experiments in western Kenya with eave tubes, a household protection product that leverages the natural behaviour of host-seeking malaria mosquitoes. METHODS: Semi-field experiments were conducted in two screen-houses. In both of these a typical western Kenyan house, with mud walls and corrugated iron sheet roofing, was built. Eave tubes with bendiocarb- or deltamethrin-treated eave tube inserts were installed in the houses, and the impact on house entry of local strains of Anopheles gambiae and Anopheles arabiensis was determined. Experiments with open eave tubes (no netting) were conducted as a control and to determine house entry through eave tubes. Insecticidal activity of the inserts treated with insecticide was examined using standard 3-min exposure bioassays. RESULTS: Experiments with open eave tubes showed that a high percentage of released mosquitoes entered the house through tubes during experimental nights. When tubes were fitted with bendiocarb- or deltamethrin-treated inserts, on average 21% [95% CI 18-25%] and 39% [CI 26-51%] of An. gambiae s.s. were recaptured the following morning, respectively. This contrasts with 71% [CI 60-81%] in the treatment with open eaves and 54% [CI 47-61%] in the treatment where inserts were treated with fluorescent dye powder. For An. arabiensis recapture was 21% [CI 14-27%] and 22% [CI 18-25%], respectively, compared to 46% [CI 40-52%] and 25% [CI 15-35%] in the treatments with open tubes and fluorescent dye. CONCLUSIONS: Insecticide-treated eave tubes resulted in significant reductions in recapture rates for both malaria vector species, representing the first and promising results with this novel control tool against Kenyan malaria vectors. Further field evaluation of eave tubes under more realistic field conditions, as well as their comparison with existing approaches in terms of cost-effectiveness and community acceptance, is called for.

Bivalent Carbamates as Novel Control Agents of the Malaria Mosquito, Anopheles gambiae.

Widespread pyrethroid resistance has caused an urgent need to develop new insecticides for control of the malaria mosquito, Anopheles gambiae. Insecticide discovery efforts were directed towards the construction of bivalent inhibitors that occupy both the peripheral and catalytic sites of the mosquito acetylcholinesterase (AChE). It was hypothesized that this approach would yield a selective, high potency inhibitor that would also circumvent known catalytic site mutations (e.g. G119S) causing target site resistance. Accordingly, a series of bivalent phthalimide-pyrazole carbamates were prepared having an alkyl chain linker of varying length, along with other modifications. The most active compound was (1-(3-(1,3-dioxoisoindolin-2-yl)propyl)-1H-pyrazol-4-yl methylcarbamate, 8a), which has a chain length of three carbons, good mosquito anticholinesterase activity, and ca. 5-fold selectivity compared to human AChE. Moreover, this compound was toxic to mosquitoes by topical application (LD50 = 63 ng/female) with only 6-fold cross resistance in the Akron strain of Anopheles gambiae that showed 50- to 60-fold resistance to conventional carbamate insecticides. However, contact lethality in the WHO paper assay was disappointing. The implications of these results for design of new mosquitocides are discussed.

Difluoromethyl ketones: Potent inhibitors of wild type and carbamate-insensitive G119S mutant Anopheles gambiae acetylcholinesterase.

Malaria is a devastating disease in sub-Saharan Africa, and current vector control measures are threatened by emerging resistance mechanisms. With the goal of developing new, selective, resistance-breaking insecticides we explored α-fluorinated methyl ketones as reversible covalent inhibitors of Anopheles gambiae acetylcholinesterase (AgAChE). Trifluoromethyl ketones 5 demonstrated remarkable volatility in microtiter plate assays, but 5c,e-h exhibited potent (1-100 nM) inhibition of wild type (WT) AgAChE and weak inhibition of resistant mutant G119S mutant AgAChE. Fluoromethyl ketones 10c-i exhibited submicromolar to micromolar inhibition of WT AgAChE, but again only weakly inhibited G119S AgAChE. Interestingly, difluoromethyl ketone inhibitors 9c and 9g had single digit nanomolar inhibition of WT AgAChE, and 9g had excellent potency against G119S AgAChE. Approach to steady-state inhibition was quite slow, but after 23 h incubation an IC50 value of 25.1 ± 1.2 nM was measured. We attribute the slow, tight-binding G119S AgAChE inhibition of 9g to a balance of steric size and electrophilicity. However, toxicities of 5g, 9g, and 10g to adult A. gambiae in tarsal contact, fumigation, and injection assays were lower than expected based on WT AgAChE inhibition potency and volatility. Potential toxicity-limiting factors are discussed.

3-Oxoisoxazole-2(3H)-carboxamides and isoxazol-3-yl carbamates: Resistance-breaking acetylcholinesterase inhibitors targeting the malaria mosquito, Anopheles gambiae.

To identify potential selective and resistance-breaking mosquitocides against the African malaria vector Anopheles gambiae, we investigated the acetylcholinesterase (AChE) inhibitory and mosquitocidal properties of isoxazol-3-yl dimethylcarbamates (15), and the corresponding 3-oxoisoxazole-2(3H)-dimethylcarboxamide isomers (14). In both series, compounds were found with excellent contact toxicity to wild-type susceptible (G3) strain and multiply resistant (Akron) strain mosquitoes that carry the G119S resistance mutation of AChE. Compounds possessing good to excellent toxicity to Akron strain mosquitoes inhibit the G119S mutant of An. gambiae AChE (AgAChE) with ki values at least 10- to 600-fold higher than that of propoxur, a compound that does not kill Akron mosquitoes at the highest concentration tested. On average, inactivation of WT AgAChE by dimethylcarboxamides 14 was 10-20 fold faster than that of the corresponding isoxazol-3-yl dimethylcarbamates 15. X-ray crystallography of dimethylcarboxamide 14d provided insight into that reactivity, a finding that may explain the inhibitory power of structurally-related inhibitors of hormone-sensitive lipase. Finally, human/An. gambiae AChE inhibition selectivities of these compounds were low, suggesting the need for additional structural modification.

Carbamate and pyrethroid resistance in the akron strain of Anopheles gambiae.

Insecticide resistance in the malaria vector, Anopheles gambiae, is a serious problem, epitomized by the multi-resistant Akron strain, originally isolated in the country of Benin. Here we report resistance in this strain to pyrethroids and DDT (13-fold to 35-fold compared to the susceptible G3 strain), but surprisingly little resistance to etofenprox, a compound sometimes described as a "pseudo-pyrethroid." There was also strong resistance to topically-applied commercial carbamates (45-fold to 81-fold), except for the oximes aldicarb and methomyl. Biochemical assays showed enhanced cytochrome P450 monooxygenase and carboxylesterase activity, but not that of glutathione-S-transferase. A series of substituted α,α,α,-trifluoroacetophenone oxime methylcarbamates were evaluated for enzyme inhibition potency and toxicity against G3 and Akron mosquitoes. The compound bearing an unsubstituted phenyl ring showed the greatest toxicity to mosquitoes of both strains. Low cross resistance in Akron was retained by all analogs in the series. Kinetic analysis of acetylcholinesterase activity and its inhibition by insecticides in the G3 strain showed inactivation rate constants greater than that of propoxur, and against Akron enzyme inactivation rate constants similar to that of aldicarb. However, inactivation rate constants against recombinant human AChE were essentially identical to that of the G3 strain. Thus, the acetophenone oxime carbamates described here, though potent insecticides that control resistant Akron mosquitoes, require further structural modification to attain acceptable selectivity and human safety.

A mathematical model for mapping the insecticide resistance trend in the Anopheles gambiae mosquito population under climate variability in Africa.

The control of arthropod disease vectors using chemical insecticides is vital in combating malaria, however the increasing insecticide resistance (IR) poses a challenge. Furthermore, climate variability affects mosquito population dynamics and subsequently IR propagation. We present a mathematical model to decipher the relationship between IR in Anopheles gambiae populations and climate variability. By adapting the susceptible-infected-resistant (SIR) framework and integrating temperature and rainfall data, our model examines the connection between mosquito dynamics, IR, and climate. Model validation using field data achieved 92% accuracy, and the sensitivity of model parameters on the transmission potential of IR was elucidated (e.g. µPRCC = 0.85958, p-value < 0.001). In this study, the integration of high-resolution covariates with the SIR model had a significant impact on the spatial and temporal variation of IR among mosquito populations across Africa. Importantly, we demonstrated a clear association between climatic variability and increased IR (width = [0-3.78], α = 0.05). Regions with high IR variability, such as western Africa, also had high malaria incidences thereby corroborating the World Health Organization Malaria Report 2021. More importantly, this study seeks to bolster global malaria combat strategies by highlighting potential IR 'hotspots' for targeted intervention by National malria control programmes.

Neurotoxicology of bis(n)-tacrines on Blattella germanica and Drosophila melanogaster acetylcholinesterase.

A series of bis(n)-tacrines were used as pharmacological probes of the acetylcholinesterase (AChE) catalytic and peripheral sites of Blattella germanica and Drosophila melanogaster, which express AChE-1 and AChE-2 isoforms, respectively. In general, the potency of bis(n)-tacrines was greater in D. melanogaster AChE (DmAChE) than in B. germanica AChE (BgAChE). The change in potency with tether length was high in DmAChE and low in BgAChE, associated with 90-fold and 5.2-fold maximal potency gain, respectively, compared to the tacrine monomer. The optimal tether length for Blattella was 8 carbons and for Drosophila was 10 carbons. The two species differed by only about twofold in their sensitivity to tacrine monomer, indicating that differential potency occurred among dimeric bis(n)-tacrines due to structural differences in the peripheral site. Multiple sequence alignment and in silico homology modeling suggest that aromatic residues of DmAChE confer higher affinity binding, and the lack of same at the BgAChE peripheral site may account, at least in part, to the greater overall sensitivity of DmAChE to bis(n)-tacrines, as reflected by in vitro assay data. Topical and injection assays in cockroaches found minimal toxicity of bis(n)-tacrines. Electrophysiological studies on D. melanogaster central nervous system showed that dimeric tacrines do not readily cross the blood brain barrier, explaining the observed nonlethality to insects. Although the bis(n)-tacrines were not good insecticide candidates, the information obtained in this study should aid in the design of selective bivalent ligands targeting insect, pests, and disease vectors.

Re-engineering aryl methylcarbamates to confer high selectivity for inhibition of Anopheles gambiae versus human acetylcholinesterase.

To identify potential human-safe insecticides against the malaria mosquito we undertook an investigation of the structure-activity relationship of aryl methylcarbamates inhibitors of acetylcholinesterase (AChE). Compounds bearing a ß-branched 2-alkoxy or 2-thioalkyl group were found to possess good selectivity for inhibition of Anopheles gambiae AChE over human AChE; up to 530-fold selectivity was achieved with carbamate 11d. A 3D QSAR model is presented that is reasonably consistent with log inhibition selectivity of 34 carbamates. Toxicity of these compounds to live Anopheles gambiae was demonstrated using both tarsal contact (filter paper) and topical application protocols.

Longitudinal impact of asymptomatic malaria/HIV-1 co-infection on Plasmodium falciparum gametocyte transcript expression and transmission to Anopheles mosquitoes.

Despite significant developments towards malaria reduction, parasite transmission in the common context of HIV-1 co-infection and treatment for one or both infections has not been fully characterized. This is particularly important given that HIV-1 and malaria chemotherapies have the potential to alter gametocyte burden and mosquito infectivity. In this study, we examined 782 blood samples collected from a longitudinal cohort of 300 volunteers with asymptomatic parasitemia seeking HIV testing or treatment in the endemic region of Kisumu, Kenya, to define the impacts of HIV-1-malaria co-infection, antiretroviral therapy (ART) plus trimethoprim-sulfamethoxazole (TS) and the antimalarials artemether/lumefantrine (AL) on Plasmodium falciparum gametocyte transcript prevalence and parasite transmission to the African malaria mosquito Anopheles gambiae. Volunteers were assigned to three distinct HIV-1 groups: HIV-1 positive on treatment, HIV-1 positive newly diagnosed, and HIV-1 negative. Volunteers were monitored monthly over the course of six months. Using our highly sensitive digital droplet PCR (ddPCR) assay of three gametocyte specific transcript markers, we detected gametocyte transcripts in 51.1% of 18S positive volunteers across all study groups and time points. After correcting for multiple comparisons, the factors of HIV-1 status, time, CD4+ T-cell levels and hematocrit were not predictive of gametocyte prevalence or transmission. However, among those volunteers who were newly diagnosed with HIV-1 and malaria positive by rapid diagnostic test (RDT) at enrollment, the initiation of ART/TS and AL treatment was associated with a significant reduction in gametocyte transcript prevalence in the subsequent month when compared to HIV-1 negative volunteers treated with AL. To assess gametocyte transmissibility, volunteer blood samples were used in standard membrane feeding assays (SFMA) with laboratory-reared A. gambiae, with evidence of transmission confirmed by at least one of 25 dissected mosquitoes per sample positive for at least one midgut oocyst. HIV-1 status, CD4+ T-cell levels and hematocrit were not significantly associated with successful transmission to A. gambiae. Analysis of SMFA blood samples revealed that 50% of transmission-positive blood samples failed to test positive by Plasmodium-specific 18S ribosomal RNA quantitative PCR (qPCR) and 35% failed to test positive for any gametocyte specific transcript marker by droplet digital (ddPCR), documenting that transmission occurred in the absence of molecular parasite/gametocyte detection. Overall, these findings highlight the complexity of HIV-1 malaria co-infection and the need to further define the unpredictable role of asymptomatic parasitemia in transmission to mosquitoes.

Contemporary exploitation of natural products for arthropod-borne pathogen transmission-blocking interventions.

An integrated approach to innovatively counter the transmission of various arthropod-borne diseases to humans would benefit from strategies that sustainably limit onward passage of infective life cycle stages of pathogens and parasites to the insect vectors and vice versa. Aiming to accelerate the impetus towards a disease-free world amid the challenges posed by climate change, discovery, mindful exploitation and integration of active natural products in design of pathogen transmission-blocking interventions is of high priority. Herein, we provide a review of natural compounds endowed with blockade potential against transmissible forms of human pathogens reported in the last 2 decades from 2000 to 2021. Finally, we propose various translational strategies that can exploit these pathogen transmission-blocking natural products into design of novel and sustainable disease control interventions. In summary, tapping these compounds will potentially aid in integrated combat mission to reduce disease transmission trends.

Diversity and Molecular Characterization of Mosquitoes (Diptera: Culicidae) in Selected Ecological Regions in Kenya.

Mosquitoes play a predominant role as leading agents in the spread of vector-borne diseases and consequent mortality in humans. Despite reports on increase of new and recurrent mosquito borne-disease outbreaks such as chikungunya, dengue fever and Rift valley fever in Kenya little is known about the genetic characteristics and diversity of the vector species that have been incriminated in transmission of disease pathogens. In this study, we identified mosquito species across Kisumu, Kilifi and Nairobi Counties and determined their genetic diversity and phylogenetic relationships. PCR was used to amplify and sequence the partial cytochrome oxidase subunit 1 (CO1) gene of mosquito samples. Molecular-genetic and phylogenetic analysis of the partial cytochrome oxidase subunit 1 (CO1) gene was employed to identify their relationships with known mosquito species. Fourteen (14) haplotypes belonging to genus Aedes, nine (9) haplotypes belonging to genus Anopheles and twelve (12) haplotypes belonging to genus Culex were identified in this study. Findings from this study revealed a potentially new haplotype belonging to Anopheles genus and reported the first molecular characterization of Aedes cummnisii in Kenya. Sequence results revealed variation in mosquito species from Kilifi, Kisumu and Nairobi. Since vector competence varies greatly across species and species-complexes and is strongly associated with specific behavioural adaptations, proper species identification is important for vector control programs.

Mosquito Acetylcholinesterase as a Target for Novel Phenyl-Substituted Carbamates.

New insecticides are needed for control of disease-vectoring mosquitoes and this research evaluates the activity of new carbamate acetylcholinesterase (AChE) inhibitors. Biochemical and toxicological characterization of carbamates based on the parent structure of terbam, 3-tert-butylphenyl methylcarbamate, was performed. In vitro enzyme inhibition selectivity (Anopheles gambiae versus human) was assessed by the Ellman assay, as well as the lethality to whole insects by the World Health Organization (WHO) paper contact assay. Bromination at the phenyl C6 position increased inhibitory potency to both AChEs, whereas a 6-iodo substituent led to loss of potency, and both halogenations caused a significant reduction of mosquitocidal activity. Similarly, installation of a hexyl substituent at C6 drastically reduced inhibition of AgAChE, but showed a smaller reduction in the inhibition of hAChE. A series of 4-carboxamido analogs of the parent compound gave reduced activity against AgAChE and generally showed more activity against hAChE than AgAChE. Replacement of the 3-t-buyl group with CF3 resulted in poor anticholinesterase activity, but this compound did have measurable mosquitocidal activity. A series of methyl- and fluoro- analogs of 3-trialkylsilyl compounds were also synthesized, but unfortunately resulted in disappointing activity. Finally, a series of sulfenylated proinsecticides showed poor paper contact toxicity, but one of them had topical activity against adult female Anopheles gambiae. Overall, the analogs prepared here contributed to a better understanding of carbamate structure-activity relationships (SAR), but no new significant leads were generated.

Monooxygenase levels and knockdown resistance (kdr) allele frequencies in Anopheles gambiae and Anopheles arabiensis in Kenya.

Pyrethroid-treated bed-nets and indoor spray are important components of malaria control strategies in Kenya. Information on resistance to pyrethroid insecticides in Anopheles gambiae and An. arabiensis populations is essential to the selection of appropriate insecticides and the management of insecticide resistance. Monooxygenase activity and knockdown resistance (kdr) allele frequency are biochemical and molecular indicators of mosquito resistance to pyrethroids. This study determined baseline information on monooxygenase activity and kdr allele frequency in anopheline mosquitoes in the western region, the Great Rift Valley-central region, and the coastal region of Kenya. In total, 1,990 field-collected individuals, representing 12 An. gambiae and 22 An. arabiensis populations were analyzed. We found significant among-population variation in monooxygenase activity in An. gambiae and An. arabiensis and substantial variability among individuals within populations. Nine of 12 An. gambiae populations exhibited significantly higher average monooxygenase activity than the susceptible Kisumu reference strain. The kdr alleles (L1014S) were detected in three An. gambiae populations, and one An. arabiensis population in western Kenya, but not in the Rift Valley-central region and the coastal Kenya region. All genotypes with the kdr alleles were heterozygous, and the conservative estimation of kdr allele frequency was below 1% in these four populations. Information on monooxygenase activity and kdr allele frequency reported in this study provided baseline data for monitoring insecticide resistance changes in Kenya during the era when large-scale insecticide-treated bed-net and indoor residual spray campaigns were being implemented.