Pierre Guillet

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.

Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene.

Samples of the dengue vector mosquito Aedes aegypti (L.) (Diptera: Culicidae) were collected from 13 localities between 1995 and 1998. Two laboratory strains, Bora (French Polynesia) and AEAE, were both susceptible to DDT and permethrin; all other strains, except Larentuka (Indonesia) and Bouaké (Ivory Coast), contained individual fourth‐instar larvae resistant to permethrin. Ten strains were subjected to a range of biochemical assays. Many strains had elevated carboxylesterase activity compared to the Bora strain; this was particularly high in the Indonesian strains Salatiga and Semarang, and in the Guyane strain (Cayenne). Monooxygenase levels were increased in the Salatiga and Paea (Polynesia) strains, and reduced in the two Thai strains (Mae Kaza, Mae Kud) and the Larentuka strain. Glutathione S‐transferase activity was elevated in the Guyane strain. All other enzyme profiles were similar to the susceptible strain. The presence of both DDT and pyrethroid resistance in the Semarang, Belem (Brazil) and Long Hoa (Vietnam) strains suggested the presence of a knock‐down resistant (kdr)‐type resistance mechanism. Part of the S6 hydrophobic segment of domain II of the voltage‐gated sodium channel gene was obtained by RT‐PCR and sequenced from several insects from all 13 field strains. Four novel mutations were identified. Three strains contained identical amino acid substitutions at two positions, two strains shared a different substitution, and one strain was homozygous for a fourth alteration. The leucine to phenylalanine substitution that confers nerve insensitivity to pyrethroids in a range of other resistant insects was absent. Direct neurophysiological assays on individual larvae from three strains with these mutations demonstrated reduced nerve sensitivity to permethrin or lambda cyhalothrin inhibition compared to the susceptible strains.

Alternative insecticides: an urgent need

Most insecticides used against pests and vectors of human disease (e.g. fleas, flies and mosquitoes) are spin-offs from agrochemical research and development. The arsenal of safe and cost-effective public health insecticides is being depleted by restrictions for various reasons (e.g. insecticide resistance, unacceptable side effects and non re-registration) and the number of new products launched is dwindling. Mobilizing public resources and establishment of partnerships to support research and development of public health insecticides is crucial in the post-DDT and post-pyrethroid era.

The kdr mutation occurs in the Mopti form of Anopheles gambiae s.s. through introgression.

Anopheles gambiae s.s. is a complex of sibling taxa characterized by various paracentric inversions. In west and central Africa, where several taxa are sympatric, a kdr mutation responsible for pyrethroid resistance has been described in only one (the S taxon), suggesting an absence of gene flow between them. Following a thorough sampling, we have found a kdr mutation in another taxon (M). To establish whether this mutation is the same event or not, the large intron upstream of the kdr mutation was sequenced to find polymorphic sites in susceptible/resistant and M/S mosquitoes. The low genetic diversity found in this DNA region indicates that a local genetic sweep has recently occurred. However, some polymorphic sites were found, and it is therefore concluded that the kdr mutation in the M taxon is not an independent mutation event, and is best explained by an introgression from the S taxon. These results are discussed within the context of possible gene flow between members of An. gambiae s.s. taxa, and with the possible spread of the kdr mutation in other closely related malaria vectors of the An. gambiae complex.

Exploiting the potential of vector control for disease prevention

Although vector control has proven highly effective in preventing disease transmission, it is not being used to its full potential, thereby depriving disadvantaged populations of the benefits of well tried and tested methods. Following the discovery of synthetic residual insecticides in the 1940s, large-scale programmes succeeded in bringing many of the important vector-borne diseases under control. By the late 1960s, most vector-borne diseases--with the exception of malaria in Africa--were no longer considered to be of primary public health importance. The result was that control programmes lapsed, resources dwindled, and specialists in vector control disappeared from public health units. Within two decades, many important vector-borne diseases had re-emerged or spread to new areas. The time has come to restore vector control to its key role in the prevention of disease transmission, albeit with an increased emphasis on multiple measures, whether pesticide-based or involving environmental modification, and with a strengthened managerial and operational capacity. Integrated vector management provides a sound conceptual framework for deployment of cost-effective and sustainable methods of vector control. This approach allows for full consideration of the complex determinants of disease transmission, including local disease ecology, the role of human activity in increasing risks of disease transmission, and the socioeconomic conditions of affected communities.

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).

Insecticide Susceptibility Status of Anopheles gambiae s.l. (Diptera : Culicidae) in the Republic of Cameroon

Abstract A large-scale survey of Anopheles gambiae Giles, 1902 susceptibility to DDT, dieldrin, permethrin, and deltamethrin was conducted in the Republic of Cameroon. 15 field populations from various geographical areas were tested using World Health Organization test kits for adult mosquitoes. The laboratory Kisumu susceptible reference strain was tested as a control. Results showed that dieldrin and DDT resistance was still present in some populations, and indicated permethrin or deltamethrin resistance. Within the Anopheles gambiae complex, resistant individuals belonged to An. gambiae s.s. and An. arabiensis species. Both M and S molecular forms of An. gambiae s.s. were found resistant. In most of resistant populations, the knockdown times were 2–5-folds increased. However, none of the surviving mosquitoes was positive to the kdr “Leu-Phe” mutation using polymerase chain reaction (PCR) diagnostic test. These results likely suggested involvement of other resistance mechanism(s), such as enzyme detoxification or kdr “Leu-Ser” mutation. Researches on An. gambiae s.l. resistance should be promoted in Cameroon, to improve malaria vector control programs and to implement resistance management strategies.

The spread of the Leu-Phe kdr mutation through Anopheles gambiae complex in Burkina Faso: genetic introgression and de novo phenomena

During extensive sampling in Burkina Faso and other African countries, the Leu‐Phe mutation producing the kdr pyrethroid resistance phenotype was reported in both Anopheles gambiae ss and A. arabiensis. This mutation was widely distributed at high frequency in the molecular S form of A. gambiae while it has been observed at a very low frequency in both the molecular M form and A. arabiensis in Burkina Faso. While the mutation in the M form is inherited through an introgression from the S form, its occurrence is a new and independent mutation event in A. arabiensis. Three nucleotides in the upstream intron of the kdr mutation differentiated A. arabiensis from A. gambiae ss and these specific nucleotides were associated with kdr mutation in A. arabiensis. Ecological divergences which facilitated the spread of the kdr mutation within the complex of A. gambiae ss in West Africa, are discussed.

Long-lasting treated mosquito nets: a breakthrough in malaria prevention

Editor – Regardless of views expressed recently in the Bulletin (1–2), insecticide-treated mosquito nets are currently the only viable option to prevent malaria transmission in large parts of Africa. To be effective, mosquito nets have to be treated by dipping them in suitable insecticides at least once a year. Less than 5% of the nets currently used in Africa are properly treated or retreated. Looking for practical solutions, WHO stimulated industry to produce long-lasting insecticide-treated mosquito nets (LLNs), using new bioactive fabric technologies. Long-lasting nets are treated only once, at factory level. The insecticide can resist multiple washes and is released over time to the surface of the netting fibres. The major criteria for LLNs is that efficacy should last as long as the average lifespan of the net, i.e. 4–5 years. Compared with nets treated by conventional dipping, LLNs have several important advantages: no need for re-treatment; reduced insecticide consumption; and minimum potential environmental impact: release of insecticide in natural water bodies during washing is greatly reduced. There are two LLNs commercially available, currently being evaluated through the WHO Pesticide Evaluation Scheme (WHOPES). R&D is crucial in further improving the technology. Second-generation LLNs are now emerging with even better performance. They are major technical breakthroughs, significantly changing prospects for successful implementation of ITN programmes, especially in Africa. To protect the most vulnerable groups in Africa (children under five, pregnant women and people living below the poverty line) and to meet the objective set by the Heads of State of African countries in Abuja (60% of net coverage by 2005), it is estimated that a total of 115 million nets will be needed. To reach full coverage, ideally by the year 2007, an additional 97 million nets will be needed and maintenance of this coverage after 2007 will require 28 to 30 million nets per year. Protection of the whole population at risk of malaria in Africa would require about twice these numbers. When envisaging further development of LLNs, it is important to involve the net industry in Africa and to facilitate technology transfer. Strengthening production capacity of LLNs in Africa and further reducing the price through well-targeted public– private partnerships will be an effective way to go to scale, reduce the malaria burden, and contribute to poverty alleviation. It also offers an opportunity to African countries to develop a profitable ITN economy and to play an increasing role in …

Dosage-dependent effects of permethrin-treated nets on the behaviour of Anopheles gambiae and the selection of pyrethroid resistance

BackgroundThe evolution and spread of pyrethroid resistance in Anopheles gambiae s.s, the major malaria vector in sub-Saharan Africa, is of great concern owing to the importance of pyrethroid-treated nets in the WHO global strategy for malaria control. The impact of kdr (the main pyrethroid-resistance mechanism) on the behaviour of An. gambiae is not well understood. The objective of this study was to determine whether high or low doses of permethrin differ in their resistance-selection effects.MethodsThe effect of permethrin treatment was assessed under laboratory conditions using the tunnel test technique against susceptible, heterozygous and homozygous genotypes. Experimental huts trials were then carried out in Benin to assess the level of personal protection conferred by nets treated with a variety of permethrin concentrations and their impact on the selection for kdr allele.ResultsTunnel tests showed that nets treated with permethrin at 250 and 500 mg/m2 induced higher mortality and blood feeding reduction among susceptible and heterozygous (RS) females as compared to the lower concentration (100 mg/m2). The experimental hut trials showed that the best personal protection was achieved with the highest permethrin concentration (1,000 mg/m2). Mosquito genotyping revealed a non-linear relationship in the survival of kdr susceptible and resistant genotypes with permethrin dosage. Higher dosages (≥250 mg/m2) killed more efficiently the RS genotypes than did lower dosages (50 and 100 mg/m2).ConclusionThis study showed that nets treated with high permethrin concentrations provided better blood feeding prevention against pyrethroid-resistant An. gambiae than did lower concentrations. Permethrin-treated nets seem unlikely to select for pyrethroid resistance in areas where the kdr mutation is rare and present mainly in heterozygous form.