Rapid and non-destructive identification of Anopheles gambiae and Anopheles arabiensis mosquito species using Raman spectroscopy via machine learning classification models.

BACKGROUND: Identification of malaria vectors is an important exercise that can result in the deployment of targeted control measures and monitoring the susceptibility of the vectors to control strategies. Although known to possess distinct biting behaviours and habitats, the African malaria vectors Anopheles gambiae and Anopheles arabiensis are morphologically indistinguishable and are known to be discriminated by molecular techniques. In this paper, Raman spectroscopy is proposed to complement the tedious and time-consuming Polymerase Chain Reaction (PCR) method for the rapid screening of mosquito identity. METHODS: A dispersive Raman microscope was used to record spectra from the legs (femurs and tibiae) of fresh anaesthetized laboratory-bred mosquitoes. The scattered Raman intensity signal peaks observed were predominantly centered at approximately 1400 cm-1, 1590 cm-1, and 2067 cm-1. These peaks, which are characteristic signatures of melanin pigment found in the insect cuticle, were important in the discrimination of the two mosquito species. Principal Component Analysis (PCA) was used for dimension reduction. Four classification models were built using the following techniques: Linear Discriminant Analysis (LDA), Logistic Regression (LR), Quadratic Discriminant Analysis (QDA), and Quadratic Support Vector Machine (QSVM). RESULTS: PCA extracted twenty-one features accounting for 95% of the variation in the data. Using the twenty-one principal components, LDA, LR, QDA, and QSVM discriminated and classified the two cryptic species with 86%, 85%, 89%, and 93% accuracy, respectively on cross-validation and 79%, 82%, 81% and 93% respectively on the test data set. CONCLUSION: Raman spectroscopy in combination with machine learning tools is an effective, rapid and non-destructive method for discriminating and classifying two cryptic mosquito species, Anopheles gambiae and Anopheles arabiensis belonging to the Anopheles gambiae complex.

Health care provider practices in diagnosis and treatment of malaria in rural communities in Kisumu County, Kenya.

BACKGROUND: Accurate malaria diagnosis and appropriate treatment at local health facilities are critical to reducing morbidity and human reservoir of infectious gametocytes. The current study assessed the accuracy of malaria diagnosis and treatment practices in three health care facilities in rural western Kenya. METHODS: The accuracy of malaria detection and treatment recommended compliance was monitored in two public and one private hospital from November 2019 through March 2020. Blood smears from febrile patients were examined by hospital laboratory technicians and re-examined by an expert microscopists thereafter subjected to real-time polymerase chain reaction (RT-PCR) for quality assurance. In addition, blood smears from patients diagnosed with malaria rapid diagnostic tests (RDT) and presumptively treated with anti-malarial were re-examined by an expert microscopist. RESULTS: A total of 1131 febrile outpatients were assessed for slide positivity (936), RDT (126) and presumptive diagnosis (69). The overall positivity rate for Plasmodium falciparum was 28% (257/936). The odds of slide positivity was higher in public hospitals, 30% (186/624, OR:1.44, 95% CI = 1.05-1.98, p < 0.05) than the private hospital 23% (71/312, OR:0.69, 95% CI = 0.51-0.95, p < 0.05). Anti-malarial treatment was dispensed more at public hospitals (95.2%, 177/186) than the private hospital (78.9%, 56/71, p < 0.0001). Inappropriate anti-malarial treatment, i.e. artemether-lumefantrine given to blood smear negative patients was higher at public hospitals (14.6%, 64/438) than the private hospital (7.1%, 17/241) (p = 0.004). RDT was the most sensitive (73.8%, 95% CI = 39.5-57.4) and specific (89.2%, 95% CI = 78.5-95.2) followed by hospital microscopy (sensitivity 47.6%, 95% CI = 38.2-57.1) and specificity (86.7%, 95% CI = 80.8-91.0). Presumptive diagnosis had the lowest sensitivity (25.7%, 95% CI = 13.1-43.6) and specificity (75.0%, 95% CI = 50.6-90.4). RDT had the highest non-treatment of negatives [98.3% (57/58)] while hospital microscopy had the lowest [77.3% (116/150)]. Health facilities misdiagnosis was at 27.9% (77/276). PCR confirmed 5.2% (4/23) of the 77 misdiagnosed cases as false positive and 68.5% (37/54) as false negative. CONCLUSIONS: The disparity in malaria diagnosis at health facilities with many slide positives reported as negatives and high presumptive treatment of slide negative cases, necessitates augmenting microscopic with RDTs and calls for Ministry of Health strengthening supportive infrastructure to be in compliance with treatment guidelines of Test, Treat, and Track to improve malaria case management.

Hyper-prevalence of submicroscopic Plasmodium falciparum infections in a rural area of western Kenya with declining malaria cases.

BACKGROUND: The gold standard for diagnosing Plasmodium falciparum infection is microscopic examination of Giemsa-stained peripheral blood smears. The effectiveness of this procedure for infection surveillance and malaria control may be limited by a relatively high parasitaemia detection threshold. Persons with microscopically undetectable infections may go untreated, contributing to ongoing transmission to mosquito vectors. The purpose of this study was to determine the magnitude and determinants of undiagnosed submicroscopic P. falciparum infections in a rural area of western Kenya. METHODS: A health facility-based survey was conducted, and 367 patients seeking treatment for symptoms consistent with uncomplicated malaria in Homa Bay County were enrolled. The frequency of submicroscopic P. falciparum infection was measured by comparing the prevalence of infection based on light microscopic inspection of thick blood smears versus real-time polymerase chain reaction (RT-PCR) targeting P. falciparum 18S rRNA gene. Long-lasting insecticidal net (LLIN) use, participation in nocturnal outdoor activities, and gender were considered as potential determinants of submicroscopic infections. RESULTS: Microscopic inspection of blood smears was positive for asexual P. falciparum parasites in 14.7% (54/367) of cases. All of these samples were confirmed by RT-PCR. 35.8% (112/313) of blood smear negative cases were positive by RT-PCR, i.e., submicroscopic infection, resulting in an overall prevalence by RT-PCR alone of 45.2% compared to 14.7% for blood smear alone. Females had a higher prevalence of submicroscopic infections (35.6% or 72 out of 202 individuals, 95% CI 28.9-42.3) compared to males (24.2%, 40 of 165 individuals, 95% CI 17.6-30.8). The risk of submicroscopic infections in LLIN users was about half that of non-LLIN users (OR = 0.59). There was no difference in the prevalence of submicroscopic infections of study participants who were active in nocturnal outdoor activities versus those who were not active (OR = 0.91). Patients who participated in nocturnal outdoor activities and use LLINs while indoors had a slightly higher risk of submicroscopic infection than those who did not use LLINs (OR = 1.48). CONCLUSION: Microscopic inspection of blood smears from persons with malaria symptoms for asexual stage P. falciparum should be supplemented by more sensitive diagnostic tests in order to reduce ongoing transmission of P. falciparum parasites to local mosquito vectors.

The effect of mass mosquito trapping on malaria transmission and disease burden (SolarMal): a stepped-wedge cluster-randomised trial.

BACKGROUND: Odour baits can attract host-seeking Anopheles mosquitoes indoors and outdoors. We assessed the effects of mass deployment of odour-baited traps on malaria transmission and disease burden. METHODS: We installed solar-powered odour-baited mosquito trapping systems (SMoTS) to households on Rusinga Island, Lake Victoria, western Kenya (mean population 24 879), in a stepped-wedge cluster-randomised trial. All residents in the completed health and demographic surveillance system were eligible to participate. We used the travelling salesman algorithm to assign all households to a cluster (50 or 51 geographically contiguous households); nine contiguous clusters formed a metacluster. Initially, no cluster had SMoTS (non-intervened). During the course of the intervention roll-out SMoTS were gradually installed cluster by cluster until all clusters had SMoTS installed (intervened). We generated 27 cluster randomisations, with the cluster as unit of randomisation, to establish the order to install the traps in the clusters until all had a SMoTS installed. Field workers and participants were not masked to group allocation. The primary outcome of clinical malaria was monitored through repeated household visits covering the entire population, once before roll-out (baseline) and five times throughout the 2-year roll-out. We measured clinical malaria as fever plus a positive result with a rapid diagnostic test. The SolarMal project was registered on the Dutch Trial Register (NTR 3496). FINDINGS: We enrolled 34 041 participants between April 25, 2012, and March 23, 2015, to 81 clusters and nine metaclusters. 4358 households were provided with SMoTS during roll-out between June 3, 2013, and May 16, 2015. 23 clinical malaria episodes were recorded in intervened clusters and 33 episodes in non-intervened clusters (adjusted effectiveness 40·8% [95% CI -172·8 to 87·1], p=0·5) during the roll-out. Malaria prevalence measured by rapid diagnostic test was 29·8% (95% CI 20·9-38·0) lower in SMoTS clusters (prevalence 23·7%; 1552 of 6550 people) than in non-intervened clusters (prevalence 34·5%; 2002 of 5795 people). INTERPRETATION: The unexpectedly low clinical incidence of malaria during roll-out led to an imprecise estimate of effectiveness from the clinical incidence data. The substantial effect on malaria prevalence is explained by reduction in densities of Anopheles funestus. Odour-baited traps might be an effective malaria intervention. FUNDING: COmON Foundation.

2-Butanone as a carbon dioxide mimic in attractant blends for the Afrotropical malaria mosquitoes Anopheles gambiae and Anopheles funestus.

BACKGROUND: Most odour baits designed to attract host-seeking mosquitoes contain carbon dioxide (CO2), which enhances trap catches, given its role as a mosquito flight activator. However, the use of CO2 is expensive and logistically demanding for prolonged area-wide use. METHODS: This study explored the possibility of replacing organically-produced CO2 with 2-butanone in odour blends targeting host-seeking malaria mosquitoes. During semi-field and field experiments MM-X traps were baited with a human odour mimic (MB5 blend) plus CO2 or 2-butanone at varying concentrations. Unbaited traps formed a control. The attraction of Anopheles gambiae s.s., Anopheles arabiensis and Anopheles funestus to these differently baited traps was measured and mean catch sizes were compared to determine whether 2-butanone could form a viable replacement for CO2 for these target species. RESULTS: Under semi-field conditions significantly more female An. gambiae mosquitoes were attracted to a reference attractant blend (MB5 + CO2) compared to MB5 without CO2 (P < 0.001), CO2 alone (P < 0.001), or a trap without a bait (P < 0.001). Whereas MB5 + CO2 attracted significantly more mosquitoes than its variants containing MB5 plus different dilutions of 2-butanone (P = 0.001), the pure form (99.5%) and the 1.0% dilution of 2-butanone gave promising results. In the field mean indoor catches of wild female An. gambiae s.l. in traps containing MB5 + CO2 (5.07 ± 1.01) and MB5 + 99.5% 2-butanone (3.10 ± 0.65) did not differ significantly (P = 0.09). The mean indoor catches of wild female An. funestus attracted to traps containing MB5 + CO2 (3.87 ± 0.79) and MB5 + 99.5% 2-butanone (3.37 ± 0.70) were also similar (P = 0.635). Likewise, the mean outdoor catches of An. gambiae and An. funestus associated with MB5 + CO2 (1.63 ± 0.38 and 0.53 ± 0.17, respectively) and MB5 + 99.5% 2-butanone (1.33 ± 0.32 and 0.40 ± 0.14, respectively) were not significantly different (P = 0.544 and P = 0.533, respectively). CONCLUSION: These results demonstrate that 2-butanone can serve as a good replacement for CO2 in synthetic blends of attractants designed to attract host-seeking An. gambiae s.l. and An. funestus mosquitoes. This development underscores the possibility of using odour-baited traps (OBTs) for monitoring and surveillance as well as control of malaria vectors and potentially other mosquito species.

Spatially variable risk factors for malaria in a geographically heterogeneous landscape, western Kenya: an explorative study.

BACKGROUND: Large reductions in malaria transmission and mortality have been achieved over the last decade, and this has mainly been attributed to the scale-up of long-lasting insecticidal bed nets and indoor residual spraying with insecticides. Despite these gains considerable residual, spatially heterogeneous, transmission remains. To reduce transmission in these foci, researchers need to consider the local demographical, environmental and social context, and design an appropriate set of interventions. Exploring spatially variable risk factors for malaria can give insight into which human and environmental characteristics play important roles in sustaining malaria transmission. METHODS: On Rusinga Island, western Kenya, malaria infection was tested by rapid diagnostic tests during two cross-sectional surveys conducted 3 months apart in 3632 individuals from 790 households. For all households demographic data were collected by means of questionnaires. Environmental variables were derived using Quickbird satellite images. Analyses were performed on 81 project clusters constructed by a traveling salesman algorithm, each containing 50-51 households. A standard linear regression model was fitted containing multiple variables to determine how much of the spatial variation in malaria prevalence could be explained by the demographic and environmental data. Subsequently, a geographically-weighted regression (GWR) was performed assuming non-stationarity of risk factors. Special attention was taken to investigate the effect of residual spatial autocorrelation and local multicollinearity. RESULTS: Combining the data from both surveys, overall malaria prevalence was 24%. Scan statistics revealed two clusters which had significantly elevated numbers of malaria cases compared to the background prevalence across the rest of the study area. A multivariable linear model including environmental and household factors revealed that higher socioeconomic status, outdoor occupation and population density were associated with increased malaria risk. The local GWR model improved the model fit considerably and the relationship of malaria with risk factors was found to vary spatially over the island; in different areas of the island socio-economic status, outdoor occupation and population density were found to be positively or negatively associated with malaria prevalence. DISCUSSION: Identification of risk factors for malaria that vary geographically can provide insight into the local epidemiology of malaria. Examining spatially variable relationships can be a helpful tool in exploring which set of targeted interventions could locally be implemented. Supplementary malaria control may be directed at areas, which are identified as at risk. For instance, areas with many people that work outdoors at night may need more focus in terms of vector control. TRIAL REGISTRATION: Trialregister.nl NTR3496-SolarMal, registered on 20 June 2012.

Enhancing Attraction of African Malaria Vectors to a Synthetic Odor Blend.

The deployment of odor-baited tools for sampling and controlling malaria vectors is limited by a lack of potent synthetic mosquito attractants. A synthetic mixture of chemical compounds referred to as "the Mbita blend" (MB) was shown to attract as many host-seeking malaria mosquitoes as attracted to human subjects. We hypothesized that this effect could be enhanced by adding one or more attractive compounds to the blend. We tested changes in the capability of MB (ammonia + L-lactic acid + tetradecanoic acid +3-methyl-1-butanol + carbon dioxide) to attract host-seeking malaria mosquitoes by addition of selected dilutions of butyl-2-methylbutanoate (1:10,000), 2-pentadecanone (1:100), 1-dodecanol (1:10,000), and butan-1-amine (1:10,000,000). The experiments were conducted in semi-field enclosures and in a village in western Kenya. In semi-field enclosures, the attraction of Anopheles gambiae sensu stricto females to MB-baited traps was not enhanced by adding butyl-2-methylbutanoate. There was, however, an increase in the proportion of An. gambiae caught in traps containing MB augmented with the selected dilutions of butan-1-amine, 2-pentadecanone, and 1-dodecanol. When tested in the village, addition of butan-1-amine to MB enhanced catches of female An. gambiae sensu lato, An. funestus, and Culex mosquitoes. 1-Dodecanol increased attraction of An. gambiae s.l. to the MB, while addition of 2-pentadecanone improved trap catches of An. funestus and Culex mosquitoes. This study demonstrates the possibility of enhancing synthetic odor blends for trapping the malarial mosquitoes An. gambiae s.l. and An. funestus, as well as some culicine species. The findings provide promising results for the optimization and utilization of synthetic attractants for sampling and controlling major disease vectors.

Mosquito host preferences affect their response to synthetic and natural odour blends.

BACKGROUND: The anthropophilic malaria mosquito Anopheles gambiae sensu stricto (hereafter termed Anopheles gambiae) primarily takes blood meals from humans, whereas its close sibling Anopheles arabiensis is more opportunistic. Previous studies have identified several compounds that play a critical role in the odour-mediated behaviour of An. gambiae. This study determined the effect of natural and synthetic odour blends on mosquitoes with different host preferences to better understand the host-seeking behaviour of mosquitoes and the potential of synthetic odour blends for standardized monitoring. METHODS: Odour blends were initially tested for their attractiveness to An. gambiae and An. arabiensis in a semi-field system with MM-X traps baited with natural and synthetic odours. Natural host odours were collected from humans, cows and chickens. The synthetic odour blends consisted of three or five previously identified compounds released with carbon dioxide. These studies were continued under natural conditions where odour blends were tested outdoors to determine their effect on species with different host preferences. RESULTS: In the semi-field experiments, human odour attracted significantly higher numbers of both mosquito species. However, An. arabiensis was also attracted to cow and chicken odours, which confirms its opportunistic behaviour. A five-component synthetic blend was highly attractive to both mosquito species. In the field, the synthetic odour blend caught significantly more An. funestus than traps baited with human odour, while no difference was found for An. arabiensis. Catches of An. arabiensis and Culex spp. contained large numbers of blood-fed mosquitoes, mostly from cows, which indicates that these mosquitoes had fed outdoors. CONCLUSIONS: Different odour baits elicit varying responses among mosquito species. Synthetic odour blends are highly effective for trapping mosquitoes; however, not all mosquitoes respond equally to the same odour blend. Combining fermenting molasses with synthetic blends in a trap represents the most effective tool to catch blood-fed mosquitoes outside houses, which is essential for understanding outdoor malaria transmission.

Mosquito Attraction: Crucial Role of Carbon Dioxide in Formulation of a Five-Component Blend of Human-Derived Volatiles.

Behavioral responses of the malaria mosquito Anopheles coluzzii (An. gambiae sensu stricto molecular 'M form') to an expanded blend of human-derived volatiles were assessed in a dual-port olfactometer. A previously documented attractive three-component blend consisting of NH3, (S)-lactic acid, and tetradecanoic acid served as the basis for expansion. Adding 4.5% CO2 to the basic blend significantly enhanced its attractiveness. Expansion of the blend with four human-derived C4-volatiles was then assessed, both with and without CO2. Only when CO2 was offered simultaneously, did addition of a specific concentration of 3-methyl-1-butanol or 3-methyl-butanoic acid significantly enhance attraction. The functional group at the terminal C of the 3-methyl-substituted C4 compounds influenced behavioral effectiveness. In the absence of CO2, addition of three concentrations of butan-1-amine caused inhibition when added to the basic blend. In contrast, when CO2 was added, butan-1-amine added to the basic blend strongly enhanced attraction at all five concentrations tested, the lowest being 100,000 times diluted. The reversal of inhibition to attraction by adding CO2 is unique in the class Insecta. We subsequently augmented the three-component basic blend by adding both butan-1-amine and 3-methyl-1-butanol and optimizing their concentrations in the presence of CO2 in order to significantly enhance the attractiveness to An. coluzzii compared to the three- and four-component blends. This novel blend holds potential to enhance malaria vector control based on behavioral disruption.

Efficacy of neem chippings for mosquito larval control under field conditions.

BACKGROUND: An in depth understanding of mosquito breeding biology and factors regulating population sizes is fundamental for vector population control. This paper presents results from a survey of mosquito breeding habitats and the efficacy of neem chippings as a potential larvicide that can be integrated in mosquito control on Nyabondo Plateau in western Kenya. RESULTS: Six main mosquito habitat types namely artificial ponds, abandoned fish ponds, active fish ponds, open drains, temporary pools and swamps were found in Nyabondo. Early anopheline instars were mainly recovered from temporary pools, artificial ponds and abandoned fish ponds. The mosquitoes collected were Anopheles gambiae sensu lato (35%), An. coustani (46%) and Culex spp (19%). Both early and late instar larvae of anopheline and culicine mosquitoes were more abundant in the controls than in the Bti and neem treated habitats. Within treated habitats, early instar anopheline mosquitoes were recovered more from habitats provided with neem and fish compared to Bti treated habitats. All treated habitats recorded higher numbers of early instar larvae than late instars or pupae, indicating that gravid female mosquitoes still oviposited within treated habitats. CONCLUSIONS: Neem chippings are a good tool for mosquito larval source management under field conditions. However, more research needs to be done to quantify the contribution of this tool to the overall mosquito borne disease transmission.

Molasses as a source of carbon dioxide for attracting the malaria mosquitoes Anopheles gambiae and Anopheles funestus.

BACKGROUND: Most odour baits for haematophagous arthropods contain carbon dioxide (CO2). The CO2 is sourced artificially from the fermentation of refined sugar (sucrose), dry ice, pressurized gas cylinders or propane. These sources of CO2 are neither cost-effective nor sustainable for use in remote areas of sub-Saharan Africa. In this study, molasses was evaluated as a potential substrate for producing CO2 used as bait for malaria mosquitoes. METHODS: The attraction of laboratory-reared and wild Anopheles gambiae complex mosquitoes to CO2 generated from yeast-fermentation of molasses was assessed under semi-field and field conditions in western Kenya. In the field, responses of wild Anopheles funestus were also assessed. Attraction of the mosquitoes to a synthetic mosquito attractant, Mbita blend (comprising ammonia, L-lactic acid, tetradecanoic acid and 3-methyl-1-butanol) when augmented with CO2 generated from yeast fermentation of either molasses or sucrose was also investigated. RESULTS: In semi-field, the release rate of CO2 and proportion of An. gambiae mosquitoes attracted increased in tandem with an increase in the quantity of yeast-fermented molasses up to an optimal ratio of molasses and dry yeast. More An. gambiae mosquitoes were attracted to a combination of the Mbita blend plus CO2 produced from fermenting molasses than the Mbita blend plus CO2 from yeast-fermented sucrose. In the field, significantly more female An. gambiae sensu lato mosquitoes were attracted to the Mbita blend augmented with CO2 produced by fermenting 500 g of molasses compared to 250 g of sucrose or 250 g of molasses. Similarly, significantly more An. funestus, Culex and other anopheline mosquito species were attracted to the Mbita blend augmented with CO2 produced from fermenting molasses than the Mbita blend with CO2 produced from sucrose. Augmenting the Mbita blend with CO2 produced from molasses was associated with high catches of blood-fed An. gambiae s.l. and An. funestus mosquitoes. CONCLUSION: Molasses is a suitable ingredient for the replacement of sucrose as a substrate for the production of CO2 for sampling of African malaria vectors and other mosquito species. The finding of blood-fed malaria vectors in traps baited with the Mbita blend and CO2 derived from molasses provides a unique opportunity for the study of host-vector interactions.

Development and optimization of the Suna trap as a tool for mosquito monitoring and control.

BACKGROUND: Monitoring of malaria vector populations provides information about disease transmission risk, as well as measures of the effectiveness of vector control. The Suna trap is introduced and evaluated with regard to its potential as a new, standardized, odour-baited tool for mosquito monitoring and control. METHODS: Dual-choice experiments with female Anopheles gambiae sensu lato in a laboratory room and semi-field enclosure, were used to compare catch rates of odour-baited Suna traps and MM-X traps. The relative performance of the Suna trap, CDC light trap and MM-X trap as monitoring tools was assessed inside a human-occupied experimental hut in a semi-field enclosure. Use of the Suna trap as a tool to prevent mosquito house entry was also evaluated in the semi-field enclosure. The optimal hanging height of Suna traps was determined by placing traps at heights ranging from 15 to 105 cm above ground outside houses in western Kenya. RESULTS: In the laboratory the mean proportion of An. gambiae s.l. caught in the Suna trap was 3.2 times greater than the MM-X trap (P < 0.001), but the traps performed equally in semi-field conditions (P = 0.615). As a monitoring tool , the Suna trap outperformed an unlit CDC light trap (P < 0.001), but trap performance was equal when the CDC light trap was illuminated (P = 0.127). Suspending a Suna trap outside an experimental hut reduced entry rates by 32.8% (P < 0.001). Under field conditions, suspending the trap at 30 cm above ground resulted in the greatest catch sizes (mean 25.8 An. gambiae s.l. per trap night). CONCLUSIONS: The performance of the Suna trap equals that of the CDC light trap and MM-X trap when used to sample An. gambiae inside a human-occupied house under semi-field conditions. The trap is effective in sampling mosquitoes outside houses in the field, and the use of a synthetic blend of attractants negates the requirement of a human bait. Hanging a Suna trap outside a house can reduce An. gambiae house entry and its use as a novel tool for reducing malaria transmission risk will be evaluated in peri-domestic settings in sub-Saharan Africa.

Ecology and behavior of Anopheles arabiensis in relation to agricultural practices in central Kenya.

Ecological changes associated with anthropogenic ecosystem disturbances can influence human risk of exposure to malaria and other vector-borne infectious diseases. This study in Mwea, Kenya, investigated the pattern of insecticide use in irrigated and nonirrigated agroecosystems and association with the density, survival, and blood-feeding behavior of the malaria vector Anopheles arabiensis. The parity rates of adult An. arabiensis from randomly selected houses were determined by examining their ovaries for tracheal distension, and polymerase chain reaction was used to identify the host blood meals. In addition, structured questionnaires were used to generate data on insecticide use. Anopheles arabiensis densities were highest in irrigated rice agroecosystems, intermediate in irrigated French beans agroecosystems, and lowest in the nonirrigated agroecosystem. Anopheles arabiensis adult survivorship was significantly lower in irrigated rice agroecosystems than in irrigated French beans agroecosystems. The human blood index (HBI) was significantly higher in the nonirrigated agroecosystem compared to irrigated agroecosystems. Moreover, there was marked variation in HBI among villages in irrigated agroecosystems with significantly lower HBI in Kangichiri and Mathangauta compared to Kiuria, Karima, and Kangai. The proportion of mosquitoes with mixed blood meals varied among villages ranging from 0.25 in Kangichiri to 0.83 in Kiuria. Sumithion, dimethoate, and alpha cypermethrin were the most commonly used insecticides. The 1st was used mostly in irrigated rice agroecosystems, and the last 2 were used mostly in irrigated French beans agroecosystems. These findings indicate that agricultural practices may influence the ecology and behavior of malaria vectors and ultimately the risk of malaria transmission.

A novel synthetic odorant blend for trapping of malaria and other African mosquito species.

Estimating the biting fraction of mosquitoes is of critical importance for risk assessment of malaria transmission. Here, we present a novel odor-based tool that has been rigorously assessed in semi-field assays and traditional African villages for estimating the number of mosquitoes that enter houses in search of a blood meal. A standard synthetic blend (SB) consisting of ammonia, (S)-lactic acid, tetradecanoic acid, and carbon dioxide was complemented with isovaleric acid, 4,5 dimethylthiazole, 2-methyl-1-butanol, and 3-methyl-1-butanol in various combinations and concentrations, and tested for attractiveness to the malaria mosquito Anopheles gambiae. Compounds were released through low density polyethylene (LDPE) material or from nylon strips (nylon). Studies were done in a semi-field facility and two traditional villages in western Kenya. The alcohol 3-methyl-1-butanol significantly increased the attraction of SB. The other compounds proved less effective or inhibitory. Tested in a village, 3-methyl-1-butanol, released from LDPE, increased the attraction of SB. Further studies showed a significantly enhanced attraction of adding 3-methyl-1-butanol to SB compared to previously-published attractive blends both under semi-field and village conditions. Other mosquito species with relevance for public health were collected with this blend in significantly higher numbers as well. These results demonstrate the advent of a novel, reliable odor-based sampling tool for the collection of malaria and other mosquitoes. The advantage of this odor-based tool over existing mosquito sampling tools is its reproducibility, objectiveness, and relatively low cost compared to current standards of CDC light traps or the human landing catch.

Integrated mosquito larval source management reduces larval numbers in two highland villages in western Kenya.

BACKGROUND: In western Kenya, malaria remains one of the major health problems and its control remains an important public health measure. Malaria control is by either use of drugs to treat patients infected with malaria parasites or by controlling the vectors. Vector control may target the free living adult or aquatic (larval) stages of mosquito. The most commonly applied control strategies target indoor resting mosquitoes. However, because mosquitoes spend a considerable time in water, targeting the aquatic stages can complement well with existing adult control measures. METHODS: Larval source management (LSM) of malaria vectors was examined in two villages i.e. Fort Ternan and Lunyerere, with the aim of testing strategies that can easily be accessed by the affected communities. Intervention strategies applied include environmental management through source reduction (drainage of canals, land levelling or by filling ditches with soil), habitat manipulation (by provision of shading from arrow root plant), application of Bacillus thuringiensis var israelensis (Bti) and the use of predatory fish, Gambusia affinis. The abundance of immature stages of Anopheles and Culex within intervention habitats was compared to that within non-intervention habitats. RESULTS: The findings show that in Fort Ternan no significant differences were observed in the abundance of Anopheles early and late instars between intervention and non-intervention habitats. In Lunyerere, the abundance of Anopheles early instars was fifty five times more likely to be present within non-intervention habitats than in habitats under drainage. No differences in early instars abundance were observed between non-intervention and habitats applied with Bti. However, late instars had 89 % and 91 % chance of being sampled from non-intervention rather than habitats under drainage and those applied with Bti respectively. CONCLUSION: Most of these interventions were applied in habitats that arose due to human activities. Involvement of community members in control programs would be beneficial in the long term once they understand the role they play in malaria transmission. Apart from the need for communities to be educated on their role in malaria transmission, there is a need to develop and test strategies that can easily be accessed and hence be used by the affected communities. The proposed LSM strategies target outdoor immature mosquitoes and hence can complement well with control measures that target indoor resting vectors. Therefore inclusion of LSM in Integrated Vector Management (IVM) program would be beneficial.

Behavioral responses of pyrethroid resistant and susceptible Anopheles gambiae mosquitoes to insecticide treated bed net.

BACKGROUND: Long-lasting insecticidal nets are an effective tool in reducing malaria transmission. However, with increasing insecticide resistance little is known about how physiologically resistant malaria vectors behave around a human-occupied bed net, despite their importance in malaria transmission. We used the Mbita bednet trap to assess the host-seeking behavior of insecticide-resistant Anopheles gambiae mosquitoes under semi-field conditions. The trap incorporates a mosquito netting panel which acts as a mechanical barrier that prevents host-seeking mosquitoes from reaching the human host baiting the trap. METHODS: Susceptible and pyrethroid-resistant colonies of female Anopheles gambiae mosquitoes aged 3-5 days old were used in this study. The laboratory-bred mosquitoes were color-marked with fluorescent powders and released inside a semi-field environment where a human subject slept inside a bednet trap erected in a traditional African hut. The netting panel inside the trap was either untreated (control) or deltamethrin-impregnated. The mosquitoes were released outside the hut. Only female mosquitoes were used. A window exit trap was installed on the hut to catch mosquitoes exiting the hut. A prokopack aspirator was used to collect indoor and outdoor resting mosquitoes. In addition, clay pots were placed outside the hut to collect outdoor resting mosquitoes. The F1 progeny of wild-caught mosquitoes were also used in these experiments. RESULTS: The mean number of resistant mosquitoes trapped in the deltamethrin-impregnated bed net trap was higher (mean = 50.21± 3.7) compared to susceptible counterparts (mean + 22.4 ± 1.31) (OR = 1.445; P<0.001). More susceptible mosquitoes were trapped in an untreated (mean = 51.9 ± 3.6) compared to a deltamethrin-treated bed net trap (mean = 22.4 ± 1.3) (OR = 2.65; P<0.001). Resistant mosquitoes were less likely to exit the house when a treated bed net was present compared to the susceptible mosquitoes. The number of susceptible mosquitoes caught resting outdoors (mean + 28.6 ± 2.22) when a treated bed net was hanged was higher than when untreated bednet was present inside the hut (mean = 4.6 ± 0.74). The susceptible females were 2.3 times more likely to stay outdoors away from the treated bed net (OR = 2.25; 95% CI = [1.7-2.9]; P<0.001). CONCLUSION: The results show that deltamethrin-treatment of netting panels inside the bednet trap did not alter the host-seeking behavior of insecticide-resistant female An. gambiae mosquitoes. On the contrary, susceptible females exited the hut and remained outdoors when a treated net was used. However, further investigations of the behavior of resistant mosquitoes under natural conditions should be undertaken to confirm these observations and improve the current intervention which are threatened by insecticide resistance and altered vector behavior.

Rare Alleles and Signatures of Selection on the Immunodominant Domains of Pfs230 and Pfs48/45 in Malaria Parasites From Western Kenya.

Background: Malaria elimination and eradication efforts can be advanced by including transmission-blocking or reducing vaccines (TBVs) alongside existing interventions. Key transmission-blocking vaccine candidates, such as Pfs230 domain one and Pfs48/45 domain 3, should be genetically stable to avoid developing ineffective vaccines due to antigenic polymorphisms. We evaluated genetic polymorphism and temporal stability of Pfs230 domain one and Pfs48/45 domain three in Plasmodium falciparum parasites from western Kenya. Methods: Dry blood spots on filter paper were collected from febrile malaria patients reporting to community health facilities in endemic areas of Homa Bay and Kisumu Counties and an epidemic-prone area of Kisii County in 2018 and 2019. Plasmodium speciation was performed using eluted DNA and real-time PCR. Amplification of the target domains of the two Pfs genes was performed on P. falciparum positive samples. We sequenced Pfs230 domain one on 156 clinical isolates and Pfs48/45 domain three on 118 clinical isolates to infer the levels of genetic variability, signatures of selection, genetic diversity indices and perform other evolutionary analyses. Results: Pfs230 domain one had low nucleotide diversity (π = 0.15 × 10-2) with slight variation per study site. Six polymorphic sites with nonsynonymous mutations and eight haplotypes were discovered. I539T was a novel variant, whereas G605S was nearing fixation. Pfs48/45 domain three had a low π (0.063 × 10-2), high conservation index, and three segregating sites, resulting in nonsynonymous mutation and four haplotypes. Some loci of Pfs230 D1 were in positive or negative linkage disequilibrium, had negative or positive selection signatures, and others (1813, 1955) and (1813, 1983) had a history of recombination. Mutated loci pairs in Pfs48/45 domain three had negative linkage disequilibrium, and some had negative and positive Tajima's D values with no history of recombination events. Conclusion: The two transmission blocking vaccine candidates have low nucleotide diversity, a small number of zone-specific variants, high nucleotide conservation index, and high frequency of rare alleles. With the near fixation a polymorphic site and the proximity of mutated codons to antibody binding epitopes, it will be necessary to continue monitoring sequence modifications of these domains when designing TBVs that include Pfs230 and Pfs48/45 antigens.

Signatures of selection and drivers for novel mutation on transmission-blocking vaccine candidate Pfs25 gene in western Kenya.

BACKGROUND: Leading transmission-blocking vaccine candidates such as Plasmodium falciparum surface protein 25 (Pfs25 gene) may undergo antigenic alterations which may render them ineffective or allele-specific. This study examines the level of genetic diversity, signature of selection and drivers of Pfs25 polymorphisms of parasites population in regions of western Kenya with varying malaria transmission intensities. METHODS: Dry blood spots (DBS) were collected in 2018 and 2019 from febrile outpatients with malaria at health facilities in malaria-endemic areas of Homa Bay, Kisumu (Chulaimbo) and the epidemic-prone highland area of Kisii. Parasites DNA were extracted from DBS using Chelex method. Species identification was performed using real-time PCR. The 460 base pairs (domains 1-4) of the Pfs25 were amplified and sequenced for a total of 180 P. falciparum-infected blood samples. RESULTS: Nine of ten polymorphic sites were identified for the first time. Overall, Pfs25 exhibited low nucleotide diversity (0.04×10-2) and low mutation frequencies (1.3% to 7.7%). Chulaimbo had the highest frequency (15.4%) of mutated sites followed by Kisii (6.7%) and Homa Bay (5.1%). Neutrality tests of Pfs25 variations showed significant negative values of Tajima's D (-2.15, p<0.01) and Fu's F (-10.91, p<0.001) statistics tests. Three loci pairs (123, 372), (364, 428) and (390, 394) were detected to be under linkage disequilibrium and none had history of recombination. These results suggested that purifying selection and inbreeding might be the drivers of the observed variation in Pfs25. CONCLUSION: Given the low level of nucleotide diversity, it is unlikely that a Pfs25 antigen-based vaccine would be affected by antigenic variations. However, continued monitoring of Pfs25 immunogenic domain 3 for possible variants that might impact vaccine antibody binding is warranted.

Impact of Environmental Modifications on the Ecology, Epidemiology, and Pathogenesis of Plasmodium falciparum and Plasmodium vivax Malaria in East Africa.

Food insecurity, recurrent famine, and poverty threaten the health of millions of African residents. Construction of dams and rural irrigation schemes is key to solving these problems. The sub-Saharan Africa International Center of Excellence for Malaria Research addresses major knowledge gaps and challenges in Plasmodium falciparum and Plasmodium vivax malaria control and elimination in malaria-endemic areas of Kenya and Ethiopia where major investments in water resource development are taking place. This article highlights progress of the International Center of Excellence for Malaria Research in malaria vector ecology and behavior, epidemiology, and pathogenesis since its inception in 2017. Studies conducted in four field sites in Kenya and Ethiopia show that dams and irrigation increased the abundance, stability, and productivity of larval habitats, resulting in increased malaria transmission and a greater disease burden. These field studies, together with hydrological and malaria transmission modeling, enhance the ability to predict the impact of water resource development projects on vector larval ecology and malaria risks, thereby facilitating the development of optimal water and environmental management practices in the context of malaria control efforts. Intersectoral collaborations and community engagement are crucial to develop and implement cost-effective malaria control strategies that meet food security needs while controlling malaria burden in local communities.

Improvement of a synthetic lure for Anopheles gambiae using compounds produced by human skin microbiota.

BACKGROUND: Anopheles gambiae sensu stricto is considered to be highly anthropophilic and volatiles of human origin provide essential cues during its host-seeking behaviour. A synthetic blend of three human-derived volatiles, ammonia, lactic acid and tetradecanoic acid, attracts A. gambiae. In addition, volatiles produced by human skin bacteria are attractive to this mosquito species. The purpose of the current study was to test the effect of ten compounds present in the headspace of human bacteria on the host-seeking process of A. gambiae. The effect of each of the ten compounds on the attractiveness of a basic blend of ammonia, lactic and tetradecanoic acid to A. gambiae was examined. METHODS: The host-seeking response of A. gambiae was evaluated in a laboratory set-up using a dual-port olfactometer and in a semi-field facility in Kenya using MM-X traps. Odorants were released from LDPE sachets and placed inside the olfactometer as well as in the MM-X traps. Carbon dioxide was added in the semi-field experiments, provided from pressurized cylinders or fermenting yeast. RESULTS: The olfactometer and semi-field set-up allowed for high-throughput testing of the compounds in blends and in multiple concentrations. Compounds with an attractive or inhibitory effect were identified in both bioassays. 3-Methyl-1-butanol was the best attractant in both set-ups and increased the attractiveness of the basic blend up to three times. 2-Phenylethanol reduced the attractiveness of the basic blend in both bioassays by more than 50%. CONCLUSIONS: Identification of volatiles released by human skin bacteria led to the discovery of compounds that have an impact on the host-seeking behaviour of A. gambiae. 3-Methyl-1-butanol may be used to increase mosquito trap catches, whereas 2-phenylethanol has potential as a spatial repellent. These two compounds could be applied in push-pull strategies to reduce mosquito numbers in malaria endemic areas.