Pyrethroid-impregnated Bednets Research Articles

Behavioral interactions of bed bugs with long-lasting pyrethroid-treated bed nets: challenges for vector control

BackgroundWidespread vector control has been essential in reducing the global incidence and prevalence of malaria, despite now stalled progress. Long-lasting insecticide-treated nets (LLINs) have historically been, and remain, one of the most commonly used vector control tools in the campaign against malaria. LLINs are effective only with proper use, adherence, retention and community adoption, which historically have relied on the successful control of secondary pests, including bed bugs. The emergence of pyrethroid-resistant bed bugs in malaria-endemic communities and failure to control infestations have been suggested to interfere with the effective use of LLINs. Therefore, the behavioral interactions of bed bugs with commonly used bed nets should be better understood.MethodsTo investigate the interactions between bed bugs (Cimex lectularius L.) and LLINs, insecticide-susceptible and pyrethroid-resistant bed bugs were challenged to pass through two commonly used LLINs in two behavioral assays, namely host (blood meal)-seeking and aggregation-seeking assays. The proportions blood-fed and aggregated bed bugs, aggregation time and mortality were quantified and analyzed in different bed bug life stages.ResultsOverall, both the insecticide-susceptible bed bugs and highly resistant bed bugs showed a varying ability to pass through LLINs based on treatment status and net design. Deltamethrin-treated nets significantly impeded both feeding and aggregation by the susceptible bed bugs. While none of the tested LLINs significantly impeded feeding (passage of unfed bed bugs through the nets) of the pyrethroid-resistant bed bugs, the untreated bed net, which has small mesh holes, impeded passage of fed bed bugs. Mortality was only seen in the susceptible bed bugs, with significantly higher mortality on deltamethrin-treated nets (63.5 ± 10.7%) than on permethrin-treated nets (2.0 ± 0.9%).ConclusionsCommonly used new LLINs failed to prevent the passage of susceptible and pyrethroid-resistant bed bugs in host- and aggregation-seeking bioassays. The overall low and variable mortality observed in susceptible bed bugs during both assays highlighted the potential of LLINs to impose strong selection pressure for the evolution of pyrethroid resistance. Already, the failure to control bed bug infestations has been implicated as a contributing factor to the abandonment or misuse of LLINs. For the first time to our knowledge, we have shown the potential of LLINs in selecting for resistant secondary pest populations and so their potential role in stalling malaria control programs should be further investigated.Graphical The emergence of pyrethroid-resistant bed bugs in malaria-endemic communities may interfere with the effective use of pyrethroid-impregnated bed nets. We assessed the interactions of two bed bug strains with commonly used bed nets using two behavioral assays, namely host (blood meal)-seeking by unfed bed bugs and aggregation-seeking by freshly fed bed bugs. These assays assessed the passage of bed bugs through various bed nets in response to host cues and aggregation stimuli, respectively. Conditioned paper is a section of file folder paper that has been exposed to bed bugs and has been impregnated with feces and aggregation pheromone; it is attractive to aggregation-seeking fed bed bugs. An unconditioned ramp is a similar section of file folder paper that allows bed bugs to traverse the bed net and gain access to a blood-meal source.

Assessing the Validity of Pyrethroid Treated Bed Net in the Curb of the Spread of Malaria Disease. A Case Study of Ijero Towhship, Ijero LGA of Ekiti State, Nigeria

This research work had shed light on assessing the effectiveness of pyrethroids impregnated treated bed nets in the curbing of the spread of Malaria disease in Ijero township in Ekiti State. This study focused on assessing efficiency of mosquito treated bed nets LLITNs, acknowledge people’s attitude towards the use of the LLITNs and to determine the resistance level of mosquitoes to pyrethroid impregnated treated bed nets. The methodology involved in this research entailed the use of descriptive study research where four objectives were constructed along with five research questions. From the total population of the area (167,632), two hundred was picked as sample size through simple random sampling in which questionnaires were distributed to the four political wards, which includes ward A,B,C and D respectively. And thirty questionnaires were also administered to the three health centres in Ijero Township. They were designed for the purpose of collecting necessary information on efficacy of pyrethroids impregnated bed nets in curbing of Malaria spread while also making use of secondary data from health institutions in Ijero Township and information gathered were analysed and presented in tables using simple percentage and also presented in graphs.

A sensory appendage protein protects malaria vectors from pyrethroids.

SummaryPyrethroid-impregnated bednets have driven significant reductions in malaria morbidity and mortality in Africa since the beginning of the century 1. The intense selection pressure exerted by bednets has precipitated widespread and escalating pyrethroid resistance in African Anopheles populations, threatening to reverse gains made by malaria control 2. Here we show that a leg-enriched sensory appendage protein, SAP2, confers pyrethroid resistance in Anopheles gambiae. SAP2 expression is elevated in insecticide-resistant populations and is further induced upon mosquito contact with pyrethroids. SAP2 silencing fully restores mosquito mortality, whilst its overexpression results in increased resistance, likely due to the high-affinity SAP2 binding to pyrethroid insecticides. Mining of genome sequence data reveals a selective sweep near the SAP2 locus in three West African countries, with the observed increase in haplotype associated SNPs mirroring increasing resistance reported in Burkina Faso. Our study identifies a new insecticide resistance mechanism that is likely highly relevant to malaria control efforts.

A qualitative study of community perception and acceptance of biological larviciding for malaria mosquito control in rural Burkina Faso

BackgroundVector and malaria parasite’s rising resistance against pyrethroid-impregnated bed nets and antimalarial drugs highlight the need for additional control measures. Larviciding against malaria vectors is experiencing a renaissance with the availability of environmentally friendly and target species-specific larvicides. In this study, we analyse the perception and acceptability of spraying surface water collections with the biological larvicide Bacillus thuringiensis israelensis in a single health district in Burkina Faso.MethodsA total of 12 focus group discussions and 12 key informant interviews were performed in 10 rural villages provided with coverage of various larvicide treatments (all breeding sites treated, the most productive breeding sites treated, and untreated control).ResultsRespondents’ knowledge about the major risk factors for malaria transmission was generally good. Most interviewees stated they performed personal protective measures against vector mosquitoes including the use of bed nets and sometimes mosquito coils and traditional repellents. The acceptance of larviciding in and around the villages was high and the majority of respondents reported a relief in mosquito nuisance and malarial episodes. There was high interest in the project and demand for future continuation.ConclusionThis study showed that larviciding interventions received positive resonance from the population. People showed a willingness to be involved and financially support the program. The positive environment with high acceptance for larviciding programs would facilitate routine implementation. An essential factor for the future success of such programs would be inclusion in regional or national malaria control guidelines.

Synergies in integrated malaria control

Synergies in integrated malaria control

Insecticide resistance monitoring of field‐collected Anopheles gambiae s.l. populations from Jinja, eastern Uganda, identifies high levels of pyrethroid resistance

Insecticide resistance in the malaria vector Anopheles gambiae s.l. (Diptera: Culicidae) threatens insecticide-based control efforts, necessitating regular monitoring. We assessed resistance in field-collected An. gambiae s.l. from Jinja, Uganda using World Health Organization (WHO) bioassays. Only An. gambiae s.s. and An. arabiensis (≈70%) were present. Female An. gambiae exhibited extremely high pyrethroid resistance (permethrin LT50 > 2 h; deltamethrin LT50 > 5 h). Female An. arabiensis were resistant to permethrin and exhibited reduced susceptibility to deltamethrin. However, while An. gambiae were DDT resistant, An. arabiensis were fully susceptible. Both species were fully susceptible to bendiocarb and fenitrothion. Kdr 1014S has increased rapidly in the Jinja population of An. gambiae s.s. and now approaches fixation (≈95%), consistent with insecticide-mediated selection, but is currently at a low frequency in An. arabiensis (0.07%). Kdr 1014F was also at a low frequency in An. gambiae. These frequencies preclude adequately-powered tests for an association with phenotypic resistance. PBO synergist bioassays resulted in near complete recovery of pyrethroid susceptibility suggesting involvement of CYP450s in resistance. A small number (0.22%) of An. gambiae s.s. ×An. arabiensis hybrids were found, suggesting the possibility of introgression of resistance alleles between species. The high levels of pyrethroid resistance encountered in Jinja threaten to reduce the efficacy of vector control programmes which rely on pyrethroid-impregnated bednets or indoor spraying of pyrethroids.

Bioassay evaluation on the efficacy of α–cypermethrin impregnated into long lasting insecticide treated nets using Anopheles stephensi

ObjectiveTo evaluate the bioefficacy of α-cypermethrin impregnated into long lasting insecticide treated nets (LLITNs-INTERCEPTOR®) against main malaria vector, Anopheles stephensi (An. stephensi). MethodsThe effectiveness of bed net impregnated with α-cypermethrin (INTERCEPTOR®) with washing was evaluated. The washing procedure and bioassay tests were carried out according to the WHO-recommended methods. Malaria vector, An. stephensi was exposed to impregnated bed net for three minutes and then mortality measured after 24 h recovery period. Knockdown was also measured according to the logarithmic times. ResultsResult of cone bioassay method showed that bioefficacy of α-cypermethrin decreased from 100% in unwashed to 15% in 20 washes. KT50 was measure as one minute in one wash and increased to 40 min in 20 washes. DiscussionFindings of this study provide guideline for malaria vector control authorities and people using pyrethroid-impregnated bed nets.

Malaria transmission and insecticide resistance of Anopheles gambiae in Libreville and Port-Gentil, Gabon

BackgroundUrban malaria is a major health priority for civilian and militaries populations. A preliminary entomologic study has been conducted in 2006-2007, in the French military camps of the two mains towns of Gabon: Libreville and Port-Gentil. The aim was to assess the malaria transmission risk for troops.MethodsMosquitoes sampled by human landing collection were identified morphologically and by molecular methods. The Plasmodium falciparum circumsporozoïte (CSP) indexes were measured by ELISA, and the entomological inoculation rates (EIR) were calculated for both areas. Molecular assessments of pyrethroid knock down (kdr) resistance and of insensitive acetylcholinesterase resistance were conducted.ResultsIn Libreville, Anopheles gambiae s.s. S form was the only specie of the An. gambiae complex present and was responsible of 9.4 bites per person per night. The circumsporozoïte index was 0.15% and the entomological inoculation rate estimated to be 1.23 infective bites during the four months period. In Port-Gentil, Anopheles melas (75.5% of catches) and An. gambiae s.s. S form (24.5%) were responsible of 58.7 bites per person per night. The CSP indexes were of 1.67% for An. gambiae s.s and 0.28% for An. melas and the EIRs were respectively of 1.8 infective bites per week and of 0.8 infective bites per week. Both kdr-w and kdr-e mutations in An. gambiae S form were found in Libreville and in Port-Gentil. Insensitive acetylcholinesterase has been detected for the first time in Gabon in Libreville.ConclusionMalaria transmission exists in both town, but with high difference in the level of risk. The co-occurrence of molecular resistances to the main families of insecticide has implications for the effectiveness of the current vector control programmes that are based on pyrethroid-impregnated bed nets.

Evaluation of bioefficacy of α-cypermethrin in long lasting impregnated net (Interceptor®) using analytical method

Objective To evaluate relationship between α-cypermethrin residues in Interceptor® nets by using HPTLC methods and mortality percentage in standard WHO's method “cone test” after repeated washings. Methods Interceptor® containing α-cypermethrin was provided by BASF Company. The washing procedure and bioassay tests were carried out according to the WHO-recommended methods. Malaria vector, An. stephensi was exposed to impregnated bed net for three minutes and then mortality measured after 24 hour recovery period. Chemical analysis was carried out in chemical laboratory by using high performance thin layer chromatography method. Results Result of cone bioassay method showed that washing reduced the efficacy of Interceptor® bed net from 100% in unwashed nets to 15% at 20 washes. After 20 washes, nets contain (61.2±2.8) mg/m 2 resulting 15% mortality in cone test. Killing activity was reduced when nets were washed. Conclusions Results will provide an essential clue for monitoring and evaluation of bioefficacy of any long lasting impregnated bed net for quality control. Findings of this study will be useful for WHO, local investigators and people who wish to use pyrethroidimpregnated bed nets for malaria vector control.

Nationwide Investigation of the Pyrethroid Susceptibility of Mosquito Larvae Collected from Used Tires in Vietnam

Pyrethroid resistance is envisioned to be a major problem for the vector control program since, at present, there are no suitable chemical substitutes for pyrethroids. Cross-resistance to knockdown agents, which are mainly used in mosquito coils and related products as spatial repellents, is the most serious concern. Since cross-resistance is a global phenomenon, we have started to monitor the distribution of mosquito resistance to pyrethroids. The first pilot study was carried out in Vietnam. We periodically drove along the national road from the north end to the Mekong Delta in Vietnam and collected mosquito larvae from used tires. Simplified susceptibility tests were performed using the fourth instar larvae of Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus. Compared with the other species, Ae. aegypti demonstrated the most prominent reduction in susceptibility. For Ae. aegypti, significant increases in the susceptibility indices with a decrease in the latitude of collection points were observed, indicating that the susceptibility of Ae. aegypti against d-allethrin was lower in the southern part, including mountainous areas, as compared to that in the northern part of Vietnam. There was a significant correlation between the susceptibility indices in Ae. aegypti and the sum of annual pyrethroid use for malaria control (1998–2002). This might explain that the use of pyrethroids as residual treatment inside houses and pyrethroid-impregnated bed nets for malaria control is attributable to low pyrethroid susceptibility in Ae. aegypti. Such insecticide treatment appeared to have been intensively administered in the interior and along the periphery of human habitation areas where, incidentally, the breeding and resting sites of Ae. aegypti are located. This might account for the strong selection pressure toward Ae. aegypti and not Ae. albopictus.

An adult-specific CYP6 P450 gene is overexpressed in a pyrethroid-resistant strain of the malaria vector, Anopheles gambiae

Many malaria control programmes are based on insecticide application as adulticides, often in the form of pyrethroid-impregnated bed nets. However, the efficacy of this control measure can be reduced by genetic changes in vector insecticide susceptibility. Pyrethroid resistance has been detected in the major African malaria vector, Anopheles gambiae, and has been attributed to a combination of target site insensitivity and increased oxidative metabolism of the insecticide, catalysed by cytochrome P450s. An adult-specific cytochrome P450 monooxygenase 6 (CYP6) P450 gene, CYP6Z1, located within a large cluster of cytochrome P450 genes in chromosome arm 3R of An. gambiae, is expressed approximately 11-fold higher in males and 4.5-fold in females from a pyrethroid-resistant strain than in a susceptible strain from the same geographical area. In both strains, CYP6Z1 expression is higher in males than females. Southern blot analysis discounted gene amplification as a cause of this overexpression. The isolation of An. gambiae cDNAs encoding cytochrome b 5 and nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH)–cytochrome P450 reductase cDNAs is also reported.

Comparative evaluation of carbosulfan- and permethrin-impregnated curtains for preventing house-entry by the malaria vector Anopheles gambiae in Burkina Faso.

Pyrethroid-impregnated bednets and curtains are widely employed to reduce the risk of malaria transmission, but pyrethroid-resistance is becoming more prevalent among malaria vector Anopheles mosquitoes (Diptera: Culicidae). As an alternative treatment for curtains, we assessed carbosulfan (a carbamate insecticide) in comparison with permethrin as the standard pyrethroid, against endophilic female mosquitoes of the Anopheles gambiae Giles complex in a village near Ouagadougou, Burkina Faso. The main criterion evaluated was the impact of curtains (hung inside windows, eaves and doorways) on the number of An. gambiae s.l. females active indoors at night. Light-traps were operated overnight (21.00-06.00 hours beside occupied untreated bednets) to sample mosquitoes in houses fitted with net curtains treated with carbosulfan 0.2 g ai/m2 or permethrin 1 g ai/m2 or untreated, compared with houses without curtains. The treated and untreated curtains significantly reduced the numbers of mosquitoes collected indoors, compared with houses without curtains. Carbosulfan-treated curtains had a highly significantly greater effect than permethrin-treated or untreated curtains, the scale of the difference being estimated as three-fold. However, there was no significant difference between the impact of untreated and permethrin-treated curtains on densities of An. gambiae s.l. trapped indoors. Samples of the An. gambiae complex comprised An. arabiensis Patton and both the S- and M-forms of An. gambiae Giles s.s. Susceptibility tests revealed some resistance to DDT and low frequencies of permethrin-resistance, insufficient to explain the poor performance of permethrin on curtains. Among survivors from the diagnostic dosage of permethrin were some specimens of all three members of the An. gambiae complex, but the kdr resistance mechanism was detected only in the S-form of An. gambiae s.s. Questions arising for further investigation include clarification of resistance mechanisms in, and foraging behaviour of, each member of the An. gambiae complex in this situation and the need to decide whether carbosulfan-treated curtains are acceptably safe for use to reduce risks of malaria transmission.

Pyrethroid resistance/susceptibility and differential urban/rural distribution of Anopheles arabiensis and An. gambiae s.s. malaria vectors in Nigeria and Ghana.

Resistance to pyrethroid insecticides and DDT caused by the kdr gene in the malaria vector Anopheles gambiae Giles s.s. (Diptera: Culicidae) has been reported in several West African countries. To test for pyrethroid resistance in two more countries, we sampled populations of the An. gambiae complex from south-western Ghana and from urban and rural localities in Ogun State, south-west Nigeria. Adult mosquitoes, reared from field-collected larvae, were exposed to the WHO-recommended discriminating dosage of exposure for 1 h to DDT 4%, deltamethrin 0.05% or permethrin 0.75% and mortality was recorded 24 h post-exposure. Susceptibility of An. gambiae s.l. to DDT was 94-100% in Ghana and 72-100% in Nigeria, indicating low levels of DDT resistance. Deltamethrin gave the highest mortality rates: 97-100% in Ghana, 95-100% in Nigeria. Ghanaian samples of An. gambiae s.l. were fully susceptible to permethrin, whereas some resistance to permethrin was detected at 4/5 Nigerian localities (percentage mortalities 75, 82, 88, 90 and 100%), with survivors including both An. arabiensis Patton and An. gambiae s.s. identified by PCR assay. Even so, the mean knockdown time was not significantly different from a susceptible reference strain, indicating absence or low frequency of kdr-type resistance. Such low levels of pyrethroid resistance are unlikely to impair the effectiveness of pyrethroid-impregnated bednets against malaria transmission. Among Nigerian samples of An. gambiae s.l., the majority from two urban localities were identified as An. arabiensis, whereas the majority from rural localities were An. gambiae s.s. These findings are consistent with those of M. Coluzzi et al. (1979). Differences of ecological distribution between molecular forms of An. gambiae s.s. were also found, with rural samples almost exclusively of the S-form, whereas the M-form predominated in urban samples. It is suggested that 'urban island' populations of An. arabiensis and of An. gambiae s.s. M-form in the rainforest belt of West Africa might be appropriate targets for elimination of these malaria vectors by the sterile insect technique.

PREVENTION OF THE TRANSMISSION OF CHAGAS’ DISEASE WITH PYRETHROID-IMPREGNATED MATERIALS

Interrupting the transmission of Chagas' disease using insecticide-treated materials could be a cost-effective option, particularly for sylvatic vectors, which enter houses at night. A randomized trial was undertaken that included all houses in two communities in regions endemic for this disease, one in Venezuela (50 houses) and one in Colombia (47 houses). After a baseline study (including a short questionnaire survey, entomologic assessment, and Chagas' disease serology), each household was randomly allocated to either the intervention group, which used pyrethroid-impregnated bed nets, or the control group, which used unimpregnated bed nets. Serologic analysis of children in the baseline study showed active transmission of Chagas' disease in the Venezuelan community (10.7% of 103 children were positive), but none in the Colombian community (0% of 100 children were positive). Vectors were sylvatic and bugs entered the houses at night in both communities. The efficacy of pyrethroids against triatomine vectors was tested in Venezuela by exposing Rhodnius prolixus to lambdacyhalothrin-impregnated fabrics and in Colombia by residual house spraying with deltamethrin. This randomized trial showed that in both countries users of impregnated bed nets were well protected from vector bites (immediate benefit). The long-term effect on the community was high vector mortality. In Venezuela, all 62 vectors detected (mainly R. robustus) died within 72 hours of contact with impregnated bed nets. In houses that used unimpregnated nets, only 24.5% (13 of 53) of the vectors died (P < 0.001). The vectors most likely came from infested palm trees and they maintained transmission of the disease in this community (28.1% of 629 R. robustus were positive for Trypanosoma cruzi). Bioassays showed that the mortality rate of R. prolixus was 100% on lambda-cyhalothrin-impregnated materials. In addition, in Colombia (the main vector was R. prolixus), the effect of repellent on vectors (driving them away from impregnated nets) was significant. Thus, users of impregnated bed nets are well protected from transmission of Chagas' disease, and vector reduction or elimination can potentially be achieved in areas infested with R. prolixus and R. robustus.

High malaria transmission intensity in a village close to Yaounde, the capital city of Cameroon.

A 2-yr longitudinal malaria study was undertaken in a suburb of Yaounde, the capital city of Cameroon, in the village of Simbock, approximately 2 km from the city limits. This study allowed assessment of malaria transmission intensity and dynamics in this region before implementation of pyrethroid impregnated bed nets through the national vector control program. Anophelines were captured on human volunteers by pyrethrum spray collections and in resting sites outdoors. Malaria vectors were Anopheles funestus Giles, Anopheles gambiae s.s. Giles (M and S forms), Anopheles moucheti Evans, and Anopheles nili Theobald. An. moucheti was the most abundant mosquito captured during the study, accounting for >54% of total anophelines caught. The annual Plasmodium falciparum Welch entomological inoculation rates measured by enzyme-linked immunosorbent assay were 277 infected bites per human for the first year and 368 for the second year. An. gambiae s.s., An. funestus, An. moucheti, and An. nili were responsible for 23.8%, 26.8%, 39.2%, and 10.2% of malaria transmission, respectively. Malaria transmission is perennial throughout the year. All these vectors were highly anthropophagous because only two out of 566 mosquitoes blood-meal tested were not taken on humans.

Olyset Net efficacy against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus after 3 years' field use in C te d'Ivoire.

Pyrethroid-impregnated bednets are advocated for personal protection against malaria vectors. To avoid the need for periodic re-treatment, it would be advantageous to have nets that retain insecticidal efficacy for years and withstand repeated washing. Such a type of commercially produced bednet with permethrin 2% incorporated in polyethylene fibres (trademark Olyset Net supplied by Sumika Life-Tech Co., Osaka, Japan) was evaluated against mosquitoes in veranda-trap huts at Yaokoffikro, near Bouaké, C te d'Ivoire, by standard WHOPES phase II procedures. Four Olyset Nets were compared with a standard untreated polyester net as control. They comprised three examples previously used in a village for over 3 years (one washed, one dirty, one very dirty) and a previously unused Olyset Net, newly unwrapped, from the same original batch. Bioassays with 3 min exposure of susceptible Anopheles gambiae Giles (Diptera: Culicidae) gave >99% mortality of female mosquitoes tested on the 'new' Olyset Net. The used Olyset Nets gave mortality rates averaging 83% for the washed net, 85% for the dirty net and 55% for the very dirty net (within 24-h following 3 min exposure). Thus, Olyset Nets were found to remain remarkably effective against susceptible An. gambiae for at least 3 years under field conditions. Wild pyrethroid-resistant populations of Culex quinquefasciatus Say and An. gambiae (savanna cytotype with 96% kdr) were assessed during June-August 1999 for their responses to sleepers protected by nets in the experimental huts. With regard to hut entry by foraging female mosquitoes, Olyset Nets showed some deterrency against An. gambiae (44% reduction by the new net, approximately 20% by the dirty nets, none by the washed net), but not against Cx. quinquefasciatus. Among mosquitoes entering the hut with untreated control net, 30-34% tried to leave (exophily) but were caught in the verandah trap. The permethrin repellency of Olyset Nets increased exophily by 19% for An. gambiae and 14% for Cx. quinquefasciatus. Blood-feeding rates were 16% An. gambiae and 35% Cx. quinquefasciatus in the hut with sleeper under the untreated net (showing considerable prevention of biting), 22-26% of both species in huts with washed or dirty used Olyset Nets (not significantly different from control), while the biting success rate of Cx. quinquefasciatus (but not kdr An. gambiae) was more than halved by the 'new' Olyset Net. Mortality rates of pyrethroid-resistant An. gambiae and Cx. quinquefasciatus from the huts were, respectively, 3% and 8% with the untreated polyester net, 27.5% and 17% with the 'new' Olyset, 15% and 17.5% with the washed Olyset, 16-25% and 17-20% with dirty old Olyset Nets. Kill differences between nets are significantly different for both An. gambiae and Cx. quinquefasciatus. Unfortunately the washed used Olyset Net showed least activity against resistant mosquitoes, despite its greatest activity against susceptible An. gambiae. In each case there was evidence that a high proportion of mosquitoes failed to feed through the net (many of them dying from starvation when they could not leave the closed hut), with indications that dirty Olyset nets enhanced this protective value.

Combined pyrethroid and carbamate 'two-in-one' treated mosquito nets: field efficacy against pyrethroid-resistant Anopheles gambiae and Culex quinquefasciatus.

A new approach is proposed in the treatment of mosquito nets, using a 'two-in-one' combination of pyrethroid and non-pyrethroid insecticides applied to different parts of bednets. The objectives are mainly to overcome certain limitations of pyrethroid-impregnated bednets currently recommended for malaria control purposes. Apart from developing alternatives to pyrethroid dependency, we sought to counteract pyrethroid irritant effects on mosquitoes (excito-repellency) and resistance to pyrethroids. The idea takes advantage of the presumed host-seeking behaviour of mosquitoes confronted by a net draped over a bed, whereby the mosquito may explore the net from the top downwards. Thus, nets could be more effective if treated on the upper part with residual non-irritant insecticide (carbamate or organophosphate) and with a pyrethroid on the lower part. Sequential exposure to different insecticides with distinct modes of action is equivalent to the use of a mixture as a potential method of managing insecticide resistance. We also intended to improve the control of nuisance mosquitoes, especially Culex quinquefasciatus Say (Diptera: Culicidae) that often survive pyrethroids, in order to encourage public compliance with use of insecticide-treated nets (ITNs). Polyester bednets were pretreated with residual pyrethroid (bifenthrin 50 mg/m2 or deltamethrin 25 mg/m2) on the lower half and with carbamate (carbosulfan 300 mg/m2) on the upper half to minimize contact with net users. Unreplicated examples of these 'two-in-one' treated nets were field-tested against wild mosquitoes, in comparison with an untreated net and bednets treated with each insecticide alone, including PermaNet wash-resistant formulation of deltamethrin 50 mg/m2. Overnight tests involved volunteers sleeping under the experimental bednets in verandah-trap huts at Yaokofikro, near Bouaké in C te d'Ivoire, where the main malaria vector Anopheles gambiae Giles, as well as Culex quinquefasciatus Say, are highly resistant to pyrethroids. Efficacy of these ITNs was assessed in the huts by four entomological criteria: deterrency and induced exophily (effects on hut entry and exit), blood-feeding and mortality rates (immediate and delayed). Overall, the best impact was achieved by the bednet treated with carbosulfan alone, followed by 'two-in-one' treatments with carbosulfan plus pyrethroid. Blood-feeding rates were 13% An. gambiae and 17% Cx. quinquefasciatus in huts with untreated nets, but only 3% with carbosulfan ITNs, 7-11% with combined ITN treatment, 6-8% An. gambiae and 12-14% Cx. quinquefasciatus with pyrethroid alone. Mosquitoes that entered the huts were killed sooner by nets with combined treatment than by pyrethroid alone. Mortality-rates in response to ITNs with carbosulfan (alone or combined with pyrethroid) were significantly greater for Cx. quinquefasciatus, but not for An. gambiae, compared to ITNs with only pyrethroid. About 20% of sleepers reported potential side-effects (headache and/or sneezing) from use of ITN treated with carbosulfan alone. Further development of this new 'two-in-one' ITN concept requires a range of investigations (choice of effective products, cost-benefit analysis, safety, etc.) leading to factory production of wash-resistant insecticidal nets treated with complementary insecticides.

Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids.

A field trial of permethrin-impregnated bednets and curtains was initiated in Western Kenya in 1990, and a strain of Anopheles gambiae showing reduced susceptibility to permethrin was colonized from this site in 1992. A leucine-phenylalanine substitution at position 1014 of the voltage-gated sodium channel is associated with resistance to permethrin and DDT in many insect species, including Anopheles gambiae from West Africa. We cloned and sequenced a partial sodium channel cDNA from the Kenyan permethrin-resistant strain and we identified an alternative substitution (leucine to serine) at the same position, which is linked to the inheritance of permethrin resistance in the F(2) progeny of genetic crosses between susceptible and resistant individuals. The diagnostic polymerase chain reaction (PCR) developed by Martinez-Torres et al. [(1998) Insect Mol Biol 7: 179-184] to detect kdr alleles in field populations of An. gambiae will not detect the Kenyan allele and hence reliance on this assay may lead to an underestimate of the prevalence of pyrethroid resistance in this species. We adapted the diagnostic PCR to detect the leucine-serine mutation and with this diagnostic we were able to demonstrate that this kdr allele was present in individuals collected from the Kenyan trial site in 1986, prior to the introduction of pyrethroid-impregnated bednets. The An. gambiae sodium channel was physically mapped to chromosome 2L, division 20C. This position corresponds to the location of a major quantitative trait locus determining resistance to permethrin in the Kenyan strain of An. gambiae.

Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s.s.

Pyrethroid-impregnated bednets are playing an increasing role for combating malaria, especially in stable malaria areas. More than 90% of the current annual malaria incidence (c. 500 million clinical cases with up to 2 million deaths) is in Africa where the major vector is Anopheles gambiae s.s. As pyrethroid resistance has been reported in this mosquito, reliable and simple techniques are urgently needed to characterize and monitor this resistance in the field. In insects, an important mechanism of pyrethroid resistance is due to a modification of the voltage-gated sodium channel protein recently shown to be associated with mutations of the para-type sodium channel gene. We demonstrate here that one of these mutations is present in certain strains of pyrethroid resistant A. gambiae s.s. and describe a PCR-based diagnostic test allowing its detection in the genome of single mosquitoes. Using this test, we found this mutation in six out of seven field samples from West Africa, its frequency being closely correlated with survival to pyrethroid exposure. This diagnostic test should bring major improvement for field monitoring of pyrethroid resistance, within the framework of malaria control programmes.

Behaviour of Anopheles albimanus in relation to pyrethroid-treated bednets.

Responses of the malaria vector Anopheles albimanus to pyrethroid impregnated bednets made of cotton or nylon, compared with untreated nets, were investigated in houses occupied by two people inside and/or outside two bednets, in coastal Chiapas, México. The pyrethroid used was lambdacyhalothrin 30 mg a.i./m2. Bioassay mortality rates of An.albimanus exposed to treated nets for 3 or 15 min, rose from 40-55% to 90-100% for nylon nets 3-19 weeks post-treatment, but were consistently lower for treated cotton nets. An.albimanus females (collected unfed on human bait) were released in houses surrounded by curtains for trapping mosquitoes that exited from the house. Floor sheets were used in and around each experimental house for retrieving any mosquitoes knocked-down and/or killed. During post-treatment assessment for 17 weeks, An.albimanus blood-feeding success rates were 23-24% with untreated nets, 14-18% with treated cotton nets and 8-15% with treated nylon nets, significantly reduced when both human baits were inside the treated bednets, but not when one or more baits were outside the treated bednet(s) within the house. Proportions of mosquitoes leaving houses < 3 h post-release were 53-59% from houses with untreated bednets versus 65-78% with treated bednets. Except in one case (when both humans were outside treated cotton nets), these increased early exit rates were significant, whether or not the human baits were inside the treated bednets indoors. Mortality rates of An.albimanus females exiting overnight (22.00-06.00 hours) averaged 15-39% from houses with treated cotton and 16-46% with treated nylon nets, very significantly greater than the control mean rates of 6-8% mortality with untreated nets. Observations on wild-caught An.albimanus females marked with fluorescent powder and released indoors revealed that few mosquitoes (3-11%) actually contacted the bednets unless both human baits remained under them - when contact rates were 22% on treated nylon, 23% on treated cotton and 42% on untreated nets (P approximately 0.05). The mean resting time was significantly longer on untreated (14.4 min) than on treated nylon (5.8 min) or cotton (9.5 min) bednets, whereas mean resting times on other surfaces indoors were 16.5-19.8 min. Proportions exiting within 2h of release were significantly more from houses with treated houses (33-35%) than with untreated nets (8%). However, mortality rates of mosquitoes that landed on treated nets were very significantly greater (90-100%) than after landing on untreated nets (10%). Thus, despite some excito-repellency, lambdacyhalothrin-impregnated bednets (especially made of nylon) proved to be effective as an alternative to house-spraying against An.albimanus.