EaveTubes for control of vector-borne diseases in Côte d'Ivoire: study protocol for a cluster randomized controlled trial.

BACKGROUND: Vector control tools, long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS), have significantly contributed to malaria prevention efforts in sub-Saharan Africa. However, insecticide resistance has seriously hampered their efficacy in recent years and new tools are essential to further progress. In2Care® EaveTubes (ETs) are an inexpensive, new resistance-breaking vector control product under World Health Organization (WHO) evaluation informed by mosquito ecology to efficiently target malaria vectors. By installing ETs in the walls of the house at the eave level that funnel the natural airflow, mosquitoes are drawn in by the same heat and odor cues that typically attract them through open eaves. Once inside an ET, mosquitoes are exposed to insecticide-treated netting placed inside the ET. The aim of this study is to test whether ETs as stand-alone tool have an effect on the epidemiology of malaria in villages where houses have been modified with the ET intervention. METHODS: A two-armed, cluster randomized controlled trial will be conducted to evaluate the effect of ETs on clinical malaria incidence in children living in Côte d'Ivoire. Thirty-four villages will be selected based on population size and the proportion of houses suitable for modification with ETs (17 treatment arms (ETs + LLINs, 17 control arms (LLINs only)). Based on the population census, 55 households per cluster with eligible children (i.e., between the ages of 6 months to 8 years old at the start of the study) will be randomly selected for recruitment into the active detection cohorts. In the treatment arm, we will enroll eligible children who reside in ET-treated houses. The intervention and control cohorts will be followed for 4 months for baseline covariate measurements and 24 months with intervention. During case detection visits, blood samples will be taken from all febrile children and tested for malaria infection with rapid diagnostic tests (RDTs). All positive clinical malaria infections will be treated. To estimate the impact of the ET on malaria vector densities, entomological measurements (indoor sampling with CDC traps) will be conducted monthly in 20 clusters (10 ET, 10 Control) in 10 randomly selected households per cluster. To estimate the infectiousness of malaria vectors, sporozoite rates will be measured in subsets of the collected mosquito samples. DISCUSSION: Findings will serve as an efficacy trial of ETs and will be submitted to the WHO Vector Control Advisory Group (VCAG) for assessment of public health value. Entomological outcomes will also be measured as proxies of malaria transmission to help develop guidelines for the evaluation of future In2Care® ETs products. TRIAL REGISTRATION: ClinicalTrials.gov NCT05736679. Registered on 10 February 2023.

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.

Eave tubes for malaria control in Africa: initial development and semi-field evaluations in Tanzania.

BACKGROUND: Presented here are a series of preliminary experiments evaluating "eave tubes"-a technology that combines house screening with a novel method of delivering insecticides for control of malaria mosquitoes. METHODS: Eave tubes were first evaluated with overnight release and recapture of mosquitoes in a screened compartment containing a hut and human sleeper. Recapture numbers were used as a proxy for overnight survival. These trials tested physical characteristics of the eave tubes (height, diameter, angle), and different active ingredients (bendiocarb, LLIN material, fungus). Eave tubes in a hut with closed eaves were also compared to an LLIN protecting a sleeper in a hut with open eaves. Eave tubes were then evaluated in a larger compartment containing a self-replicating mosquito population, vegetation, and multiple houses and cattle sheds. In this "model village", LLINs were introduced first, followed by eave tubes and associated house modifications. RESULTS: Initial testing suggested that tubes placed horizontally and at eave height had the biggest impact on mosquito recapture relative to respective controls. Comparison of active ingredients suggested roughly equivalent effects from bendiocarb, LLIN material, and fungal spores (although speed of kill was slower for fungus). The impact of treated netting on recapture rates ranged from 50 to 70 % reduction relative to controls. In subsequent experiments comparing bendiocarb-treated netting in eave tubes against a standard LLIN, the effect size was smaller but the eave tubes with closed eaves performed at least as well as the LLIN with open eaves. In the model village, introducing LLINs led to an approximate 60 % reduction in larval densities and 85 % reduction in indoor catches of host-seeking mosquitoes relative to pre-intervention values. Installing eave tubes and screening further reduced larval density (93 % relative to pre intervention values) and virtually eliminated indoor host-seeking mosquitoes. When the eave tubes and screening were removed, larval and adult catches recovered to pre-eave tube levels. CONCLUSIONS: These trials suggest that the "eave tube" package can impact overnight survival of host-seeking mosquitoes and can suppress mosquito populations, even in a complex environment. Further testing is now required to evaluate the robustness of these findings and demonstrate impact under field conditions.

Eave tubes for malaria control in Africa: an introduction.

In spite of massive progress in the control of African malaria since the turn of the century, there is a clear and recognized need for additional tools beyond long-lasting insecticide-treated bed nets (LLINs) and indoor residual spraying (IRS) of insecticides, to progress towards elimination. Moreover, widespread and intensifying insecticide resistance requires alternative control agents and delivery systems to enable development of effective insecticide resistance management strategies. This series of articles presents a novel concept for malaria vector control, the 'eave tube', which may fulfil these important criteria. From its conceptualization to laboratory and semi-field testing, to demonstration of potential for implementation, the stepwise development of this new vector control approach is described. These studies suggest eave tubes (which comprise a novel way of delivering insecticides plus screening to make the house more 'mosquito proof') could be a viable, cost-effective, and acceptable control tool for endophilic and endophagic anophelines, and possibly other (nuisance) mosquitoes. The approach could be applicable in a wide variety of housing in sub-Saharan Africa, and possibly beyond, for vectors that use the eave as their primary house entry point. The results presented in these articles were generated during an EU-FP7 funded project, the mosquito contamination device (MCD) project, which ran between 2012 and 2015. This was a collaborative project undertaken by vector biologists, product developers, modellers, materials scientists, and entrepreneurs from five different countries.

Electrostatic coating enhances bioavailability of insecticides and breaks pyrethroid resistance in mosquitoes.

Insecticide resistance poses a significant and increasing threat to the control of malaria and other mosquito-borne diseases. We present a novel method of insecticide application based on netting treated with an electrostatic coating that binds insecticidal particles through polarity. Electrostatic netting can hold small amounts of insecticides effectively and results in enhanced bioavailability upon contact by the insect. Six pyrethroid-resistant Anopheles mosquito strains from across Africa were exposed to similar concentrations of deltamethrin on electrostatic netting or a standard long-lasting deltamethrin-coated bednet (PermaNet 2.0). Standard WHO exposure bioassays showed that electrostatic netting induced significantly higher mortality rates than the PermaNet, thereby effectively breaking mosquito resistance. Electrostatic netting also induced high mortality in resistant mosquito strains when a 15-fold lower dose of deltamethrin was applied and when the exposure time was reduced to only 5 s. Because different types of particles adhere to electrostatic netting, it is also possible to apply nonpyrethroid insecticides. Three insecticide classes were effective against strains of Aedes and Culex mosquitoes, demonstrating that electrostatic netting can be used to deploy a wide range of active insecticides against all major groups of disease-transmitting mosquitoes. Promising applications include the use of electrostatic coating on walls or eave curtains and in trapping/contamination devices. We conclude that application of electrostatically adhered particles boosts the efficacy of WHO-recommended insecticides even against resistant mosquitoes. This innovative technique has potential to support the use of unconventional insecticide classes or combinations thereof, potentially offering a significant step forward in managing insecticide resistance in vector-control operations.

Development and evaluation of a novel contamination device that targets multiple life-stages of Aedes aegypti.

BACKGROUND: The increasing global threat of Dengue demands new and easily applicable vector control methods. Ovitraps provide a low-tech and inexpensive means to combat Dengue vectors. Here we describe the development and optimization process of a novel contamination device that targets multiple life-stages of the Aedes aegypti mosquito. Special focus is directed to the diverse array of control agents deployed in this trap, covering adulticidal, larvicidal and autodissemination impacts. METHODS: Different trap prototypes and their parts are described, including a floater to contaminate alighting gravid mosquitoes. The attractiveness of the trap, different odor lures and floater design were studied using fluorescent powder adhering to mosquito legs and via choice tests. We demonstrate the mosquitocidal impacts of the control agents: a combination of the larvicide pyriproxyfen and the adulticidal fungus Beauveria bassiana. The impact of pyriproxyfen was determined in free-flight dissemination experiments. The effect on larval development inside the trap and in surrounding breeding sites was measured, as well as survival impacts on recaptured adults. RESULTS: The developmental process resulted in a design that consists of a black 3 Liter water-filled container with a ring-shaped floater supporting vertically placed gauze dusted with the control agents. On average, 90% of the mosquitoes in the fluorescence experiments made contact with the gauze on the floater. Studies on attractants indicated that a yeast-containing tablet was the most attractive odor lure. Furthermore, the fungus Beauveria bassiana was able to significantly increase mortality of the free-flying adults compared to controls. Dissemination of pyriproxyfen led to >90% larval mortality in alternative breeding sites and 100% larval mortality in the trap itself, against a control mortality of around 5%. CONCLUSION: This ovitrap is a promising new tool in the battle against Dengue. It has proven to be attractive to Aedes aegypti mosquitoes and effective in contaminating these with Beauveria bassiana. Furthermore, we show that the larvicide pyriproxyfen is successfully disseminated to breeding sites close to the trap. Its low production and operating costs enable large scale deployment in Dengue-affected locations.

Development of fungal applications on netting substrates for malaria vector control.

Mosquito resistance to chemical insecticides is considered a serious threat for the sustainable use of contemporary malaria vector control methods. Fungal entomopathogens show potential as alternative biological control agents against (insecticide-resistant) anophelines. This study was designed to test whether the fungus, Beauveria bassiana, could be delivered to mosquitoes on netting materials that might be used in house screens, such as eave curtains. Tests were conducted to determine effects of formulation, application method, netting material, and nature of mosquito contact. Beauveria had a twice as high impact on Anopheles gambiae s.s. longevity when suspended in Shellsol solvent compared with Ondina oil (HR = 2.12, 95% confidence interval = 1.83-2.60, P < 0.001), and was significantly more infective when applied through spraying than dipping. Polyester and cotton bednets were the most effective substrates for mosquito infections, with highest spore viability on cotton nets. Whereas fungal impact was highest in mosquitoes that had passed through large-meshed impregnated nets, overall efficacy was equal between small- and large-meshed nets, with < or = 30-min spore contact killing >90% of mosquitoes within 10 d. Results indicate that the use of fungal spores dissolved in Shellsol and sprayed on small-meshed cotton eave curtain nets would be the most promising option for field implementation. Biological control with fungus-impregnated eave curtains could provide a means to target host-seeking mosquitoes upon house entry, and has potential for use in integrated vector management strategies, in combination with chemical vector control measures, to supplement malaria control in areas with high levels of insecticide resistance.

First report of the infection of insecticide-resistant malaria vector mosquitoes with an entomopathogenic fungus under field conditions.

BACKGROUND: Insecticide-resistant mosquitoes are compromising the ability of current mosquito control tools to control malaria vectors. A proposed new approach for mosquito control is to use entomopathogenic fungi. These fungi have been shown to be lethal to both insecticide-susceptible and insecticide-resistant mosquitoes under laboratory conditions. The goal of this study was to see whether entomopathogenic fungi could be used to infect insecticide-resistant malaria vectors under field conditions, and to see whether the virulence and viability of the fungal conidia decreased after exposure to ambient African field conditions. METHODS: This study used the fungus Beauveria bassiana to infect the insecticide-resistant malaria vector Anopheles gambiae s.s (Diptera: Culicidae) VKPER laboratory colony strain. Fungal conidia were applied to polyester netting and kept under West African field conditions for varying periods of time. The virulence of the fungal-treated netting was tested 1, 3 and 5 days after net application by exposing An. gambiae s.s. VKPER mosquitoes in WHO cone bioassays carried out under field conditions. In addition, the viability of B. bassiana conidia was measured after up to 20 days exposure to field conditions. RESULTS: The results show that B. bassiana infection caused significantly increased mortality with the daily risk of dying being increased by 2.5 × for the fungus-exposed mosquitoes compared to the control mosquitoes. However, the virulence of the B. bassiana conidia decreased with increasing time spent exposed to the field conditions, the older the treatment on the net, the lower the fungus-induced mortality rate. This is likely to be due to the climate because laboratory trials found no such decline within the same trial time period. Conidial viability also decreased with increasing exposure to the net and natural abiotic environmental conditions. After 20 days field exposure the conidial viability was 30%, but the viability of control conidia not exposed to the net or field conditions was 79%. CONCLUSIONS: This work shows promise for the use of B. bassiana fungal conidia against insecticide-resistant mosquitoes in the field, but further work is required to examine the role of environmental conditions on fungal virulence and viability with a view to eventually making the fungal conidia delivery system more able to withstand the ambient African climate.

The entomopathogenic fungus Beauveria bassiana reduces instantaneous blood feeding in wild multi-insecticide-resistant Culex quinquefasciatus mosquitoes in Benin, West Africa.

BACKGROUND: Mosquito-borne diseases are still a major health risk in many developing countries, and the emergence of multi-insecticide-resistant mosquitoes is threatening the future of vector control. Therefore, new tools that can manage resistant mosquitoes are required. Laboratory studies show that entomopathogenic fungi can kill insecticide-resistant malaria vectors but this needs to be verified in the field. METHODS: The present study investigated whether these fungi will be effective at infecting, killing and/or modifying the behaviour of wild multi-insecticide-resistant West African mosquitoes. The entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana were separately applied to white polyester window netting and used in combination with either a permethrin-treated or untreated bednet in an experimental hut trial. Untreated nets were used because we wanted to test the effect of fungus alone and in combination with an insecticide to examine any potential additive or synergistic effects. RESULTS: In total, 1125 female mosquitoes were collected during the hut trial, mainly Culex quinquefasciatus Say. Unfortunately, not enough wild Anopheles gambiae Giles were collected to allow the effect the fungi may have on this malaria vector to be analysed. None of the treatment combinations caused significantly increased mortality of Cx. quinquefasciatus when compared to the control hut. The only significant behaviour modification found was a reduction in blood feeding by Cx. quinquefasciatus, caused by the permethrin and B. bassiana treatments, although no additive effect was seen in the B. bassiana and permethrin combination treatment. Beauveria bassiana did not repel blood foraging mosquitoes either in the laboratory or field. CONCLUSIONS: This is the first time that an entomopathogenic fungus has been shown to reduce blood feeding of wild mosquitoes. This behaviour modification indicates that B. bassiana could potentially be a new mosquito control tool effective at reducing disease transmission, although further field work in areas with filariasis transmission should be carried out to verify this. In addition, work targeting malaria vector mosquitoes should be carried out to see if these mosquitoes manifest the same behaviour modification after infection with B. bassiana conidia.

Synergy in efficacy of fungal entomopathogens and permethrin against West African insecticide-resistant Anopheles gambiae mosquitoes.

BACKGROUND: Increasing incidences of insecticide resistance in malaria vectors are threatening the sustainable use of contemporary chemical vector control measures. Fungal entomopathogens provide a possible additional tool for the control of insecticide-resistant malaria mosquitoes. This study investigated the compatibility of the pyrethroid insecticide permethrin and two mosquito-pathogenic fungi, Beauveria bassiana and Metarhizium anisopliae, against a laboratory colony and field population of West African insecticide-resistant Anopheles gambiae s.s. mosquitoes. METHODOLOGY/FINDINGS: A range of fungus-insecticide combinations was used to test effects of timing and sequence of exposure. Both the laboratory-reared and field-collected mosquitoes were highly resistant to permethrin but susceptible to B. bassiana and M. anisopliae infection, inducing 100% mortality within nine days. Combinations of insecticide and fungus showed synergistic effects on mosquito survival. Fungal infection increased permethrin-induced mortality rates in wild An. gambiae s.s. mosquitoes and reciprocally, exposure to permethrin increased subsequent fungal-induced mortality rates in both colonies. Simultaneous co-exposure induced the highest mortality; up to 70.3+/-2% for a combined Beauveria and permethrin exposure within a time range of one gonotrophic cycle (4 days). CONCLUSIONS/SIGNIFICANCE: Combining fungi and permethrin induced a higher impact on mosquito survival than the use of these control agents alone. The observed synergism in efficacy shows the potential for integrated fungus-insecticide control measures to dramatically reduce malaria transmission and enable control at more moderate levels of coverage even in areas where insecticide resistance has rendered pyrethroids essentially ineffective.

Pyrethroid resistance in Anopheles gambiae leads to increased susceptibility to the entomopathogenic fungi Metarhizium anisopliae and Beauveria bassiana.

BACKGROUND: Entomopathogenic fungi are being investigated as a new mosquito control tool because insecticide resistance is preventing successful mosquito control in many countries, and new methods are required that can target insecticide-resistant malaria vectors. Although laboratory studies have previously examined the effects of entomopathogenic fungi against adult mosquitoes, most application methods used cannot be readily deployed in the field. Because the fungi are biological organisms it is important to test potential field application methods that will not adversely affect them. The two objectives of this study were to investigate any differences in fungal susceptibility between an insecticide-resistant and insecticide-susceptible strain of Anopheles gambiae sensu stricto, and to test a potential field application method with respect to the viability and virulence of two fungal species METHODS: Pieces of white polyester netting were dipped in Metarhizium anisopliae ICIPE-30 or Beauveria bassiana IMI391510 mineral oil suspensions. These were kept at 27 +/- 1 degrees C, 80 +/- 10% RH and the viability of the fungal conidia was recorded at different time points. Tube bioassays were used to infect insecticide-resistant (VKPER) and insecticide-susceptible (SKK) strains of An. gambiae s.s., and survival analysis was used to determine effects of mosquito strain, fungus species or time since fungal treatment of the net. RESULTS: The resistant VKPER strain was significantly more susceptible to fungal infection than the insecticide-susceptible SKK strain. Furthermore, B. bassiana was significantly more virulent than M. anisopliae for both mosquito strains, although this may be linked to the different viabilities of these fungal species. The viability of both fungal species decreased significantly one day after application onto polyester netting when compared to the viability of conidia remaining in suspension. CONCLUSIONS: The insecticide-resistant mosquito strain was susceptible to both species of fungus indicating that entomopathogenic fungi can be used in resistance management and integrated vector management programmes to target insecticide-resistant mosquitoes. Although fungal viability significantly decreased when applied to the netting, the effectiveness of the fungal treatment at killing mosquitoes did not significantly deteriorate. Field trials over a longer trial period need to be carried out to verify whether polyester netting is a good candidate for operational use, and to see if wild insecticide-resistant mosquitoes are as susceptible to fungal infection as the VKPER strain.

The infectivity of the entomopathogenic fungus Beauveria bassiana to insecticide-resistant and susceptible Anopheles arabiensis mosquitoes at two different temperatures.

BACKGROUND: Control of the major African malaria vector species continues to rely extensively on the application of residual insecticides through indoor house spraying or bed net impregnation. Insecticide resistance is undermining the sustainability of these control strategies. Alternatives to the currently available conventional chemical insecticides are, therefore, urgently needed. Use of fungal pathogens as biopesticides is one such possibility. However, one of the challenges to the approach is the potential influence of varied environmental conditions and target species that could affect the efficacy of a biological 'active ingredient'. An initial investigation into this was carried out to assess the susceptibility of insecticide-susceptible and resistant laboratory strains and wild-collected Anopheles arabiensis mosquitoes to infection with the fungus Beauveria bassiana under two different laboratory temperature regimes. METHODS: Insecticide susceptibility to all four classes of insecticides recommended by WHO for vector control was tested on laboratory and wild-caught An. arabiensis, using standard WHO bioassay protocols. Mosquito susceptibility to fungus infection was tested using dry spores of B. bassiana under two temperature regimes (21 +/- 1 degrees C or 25 +/- 2 degrees C) representative of indoor conditions observed in western Kenya. Cox regression analysis was used to assess the effect of fungal infection on mosquito survival and the effect of insecticide resistance status and temperature on mortality rates following fungus infection. RESULTS: Survival data showed no relationship between insecticide susceptibility and susceptibility to B. bassiana. All tested colonies showed complete susceptibility to fungal infection despite some showing high resistance levels to chemical insecticides. There was, however, a difference in fungus-induced mortality rates between temperature treatments with virulence significantly higher at 25 degrees C than 21 degrees C. Even so, because malaria parasite development is also known to slow as temperatures fall, expected reductions in malaria transmission potential due to fungal infection under the cooler conditions would still be high. CONCLUSIONS: These results provide evidence that the entomopathogenic fungus B. bassiana has potential for use as an alternative vector control tool against insecticide-resistant mosquitoes under conditions typical of indoor resting environments. Nonetheless, the observed variation in effective virulence reveals the need for further study to optimize selection of isolates, dose and use strategy in different eco-epidemiological settings.

A novel method for standardized application of fungal spore coatings for mosquito exposure bioassays.

BACKGROUND: Interest in the use of fungal entomopathogens against malaria vectors is growing. Fungal spores infect insects via the cuticle and can be applied directly on the insect to evaluate infectivity. For flying insects such as mosquitoes, however, application of fungal suspensions on resting surfaces is more realistic and representative of field settings. For this type of exposure, it is essential to apply specific amounts of fungal spores homogeneously over a surface for testing the effects of fungal dose and exposure time. Contemporary methods such as spraying or brushing spore suspensions onto substrates do not produce the uniformity and consistency that standardized laboratory assays require. Two novel fungus application methods using equipment developed in the paint industry are presented and compared. METHODS: Wired, stainless steel K-bars were tested and optimized for coating fungal spore suspensions onto paper substrates. Different solvents and substrates were evaluated. Two types of coating techniques were compared, i.e. manual and automated coating. A standardized bioassay set-up was designed for testing coated spores against malaria mosquitoes. RESULTS: K-bar coating provided consistent applications of spore layers onto paper substrates. Viscous Ondina oil formulations were not suitable and significantly reduced spore infectivity. Evaporative Shellsol T solvent dried quickly and resulted in high spore infectivity to mosquitoes. Smooth proofing papers were the most effective substrate and showed higher infectivity than cardboard substrates. Manually and mechanically applied spore coatings showed similar and reproducible effects on mosquito survival. The standardized mosquito exposure bioassay was effective and consistent in measuring effects of fungal dose and exposure time. CONCLUSIONS: K-bar coating is a simple and consistent method for applying fungal spore suspensions onto paper substrates and can produce coating layers with accurate effective spore concentrations. The mosquito bioassay was suitable for evaluating fungal infectivity and virulence, allowing optimizations of spore dose and exposure time. Use of this standardized application method will help achieve reliable results that are exchangeable between different laboratories.

Fungal infection counters insecticide resistance in African malaria mosquitoes.

The evolution of insecticide resistance in mosquitoes is threatening the effectiveness and sustainability of malaria control programs in various parts of the world. Through their unique mode of action, entomopathogenic fungi provide promising alternatives to chemical control. However, potential interactions between fungal infection and insecticide resistance, such as cross-resistance, have not been investigated. We show that insecticide-resistant Anopheles mosquitoes remain susceptible to infection with the fungus Beauveria bassiana. Four different mosquito strains with high resistance levels against pyrethroids, organochlorines, or carbamates were equally susceptible to B. bassiana infection as their baseline counterparts, showing significantly reduced mosquito survival. Moreover, fungal infection reduced the expression of resistance to the key public health insecticides permethrin and dichlorodiphenyltrichloroethane. Mosquitoes preinfected with B. bassiana or Metarhizium anisopliae showed a significant increase in mortality after insecticide exposure compared with uninfected control mosquitoes. Our results show a high potential utility of fungal biopesticides for complementing existing vector control measures and provide products for use in resistance management strategies.

African water storage pots for the delivery of the entomopathogenic fungus Metarhizium anisopliae to the malaria vectors Anopheles gambiae s.s. and Anopheles funestus.

We studied the use of African water storage pots for point source application of Metarhizium anisopliae against the malaria vectors Anopheles gambiae s.s. and An. funestus. Clay pots were shown to be attractive resting sites for male and female An. gambiae s.s. and were not repellent after impregnation with fungus. M. anisopliae was highly infective and virulent after spray application inside pots. At a dosage of 4 x 10(10) conidia/m(2), an average of 95 +/- 1.2% of An. gambiae s.s. obtained a fungal infection. A lower dosage of 1 x 10(10) conidia/m(2) infected an average of 91.5 +/- 0.6% of An. gambiae s.s. and 91.8 +/- 1.2% of An. funestus mosquitoes. Fungal infection significantly reduced mosquito longevity, as shown by differences between survival curves and LT(50) values. These pots are suitable for application of entomopathogenic fungi against malaria vectors and their potential for sustainable field implementation is discussed.

Comparison between quantitative nucleic acid sequence-based amplification, real-time reverse transcriptase PCR, and real-time PCR for quantification of Leishmania parasites.

DNA or RNA amplification methods for detection of Leishmania parasites have advantages regarding sensitivity and potential quantitative characteristics in comparison with conventional diagnostic methods but are often still not routinely applied. However, the use and application of molecular assays are increasing, but comparative studies on the performance of these different assays are lacking. The aim of this study was to compare three molecular assays for detection and quantification of Leishmania parasites in serial dilutions of parasites and in skin biopsies collected from cutaneous leishmaniasis (CL) patients in Manaus, Brazil. A serial dilution of promastigotes spiked in blood was tested in triplicate in three different runs by quantitative nucleic acid sequence-based amplification (QT-NASBA), quantitative real-time reverse transcriptase PCR (qRT-PCR), and quantitative real-time PCR (qPCR). In addition, the costs, durations, and numbers of handling steps were compared, and 84 skin biopsies from patients with suspected CL were tested. Both QT-NASBA and qRT-PCR had a detection limit of 100 parasites/ml of blood, while qPCR detected 1,000 parasites/ml. QT-NASBA had the lowest range of intra-assay variation (coefficients of variation [CV], 0.5% to 3.3%), while qPCR had the lowest range of interassay variation (CV, 0.4% to 5.3%). Furthermore, qRT-PCR had higher r2 values and amplification efficiencies than qPCR, and qPCR and qRT-PCR had faster procedures than QT-NASBA. All assays performed equally well with patient samples, with significant correlations between parasite counts. Overall, qRT-PCR is preferred over QT-NASBA and qPCR as the most optimal diagnostic assay for quantification of Leishmania parasites, since it was highly sensitive and reproducible and the procedure was relatively fast.