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      Anopheles gambiae distribution and insecticide resistance in the cities of Douala and Yaoundé (Cameroon): influence of urban agriculture and pollution

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          Abstract

          Background

          Urban malaria is becoming a major health priority across Africa. A study was undertaken to assess the importance of urban pollution and agriculture practice on the distribution and susceptibility to insecticide of malaria vectors in the two main cities in Cameroon.

          Methods

          Anopheline larval breeding sites were surveyed and water samples analysed monthly from October 2009 to December 2010. Parameters analysed included turbidity, pH, temperature, conductivity, sulfates, phosphates, nitrates, nitrites, ammonia, aluminium, alkalinity, iron, potassium, manganese, magnesium, magnesium hardness and total hardness. Characteristics of water bodies in urban areas were compared to rural areas and between urban sites. The level of susceptibility of Anopheles gambiae to 4% DDT, 0.75% permethrin, 0.05% deltamethrin, 0.1% bendiocarb and 5% malathion were compared between mosquitoes collected from polluted, non polluted and cultivated areas.

          Results

          A total of 1,546 breeding sites, 690 in Yaoundé and 856 in Douala, were sampled in the course of the study. Almost all measured parameters had a concentration of 2- to 100-fold higher in urban compare to rural breeding sites. No resistance to malathion was detected, but bendiocarb resistance was present in Yaounde. Very low mortality rates were observed following DDT or permethrin exposure, associated with high kdr frequencies. Mosquitoes collected in cultivated areas, exhibited the highest resistant levels. There was little difference in insecticide resistance or kdr allele frequency in mosquitoes collected from polluted versus non-polluted sites.

          Conclusion

          The data confirm high selection pressure on mosquitoes originating from urban areas and suggest urban agriculture rather than pollution as the major factor driving resistance to insecticide.

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          Most cited references30

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          The role of agricultural use of insecticides in resistance to pyrethroids in Anopheles gambiae s.l. in Burkina Faso.

          Agricultural use of insecticides is involved in the selection of resistance to these compounds in field populations of mosquitoes in Burkina Faso. Anopheles gambiae s.l. was resistant to permethrin and DDT in cotton-growing and urban areas, but susceptible in areas with limited insecticide selection pressure (rice fields and control areas). Nevertheless, resistance to these insecticides was observed in a village on the outskirts of the rice fields at the end of the rainy season, suggesting that the latter population of mosquitoes had migrated from the surrounding cotton villages into the rice fields. A seasonal variation of resistance observed in the cotton-growing area is related to the distribution of the molecular M and S forms of An. gambiae, since resistance to pyrethroids has so far only been reported in the S form. Pyrethroid resistance in west African An. gambiae was conferred by target site insensitivity through a knockdown resistance (kdr)-like mutation, which was present at high frequencies in mosquitoes in the cotton-growing and urban areas.
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            Multiple insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus from Benin, West Africa.

            Because free-insecticide treated net distribution is planned in Benin (West Africa) during the next few years, we investigated the type, frequency and distribution of insecticide resistance mechanisms in Anopheles gambiae and Culex quinquefasciatus mosquitoes in four localities selected on the basis of contrasting agricultural practices, use of insecticides and environment. Bioassays with WHO diagnostic test kits were carried out using pyrethroid, carbamate, organophosphate and organochlorine insecticides. An. gambiae mosquitoes were identified to species and to M or S molecular forms using PCR techniques. Molecular and biochemical assays were carried out to identify kdr and Ace.1 mutations in individual mosquitoes and to detect any increase in the activity of enzymes typically involved in insecticide metabolism (oxidase, esterase and glutathion-S-transférases). WHO diagnostic tests showed high frequency of resistance in An. gambiae and Cx. quinquefasciatus to permethrin and DDT in three areas. This was consistent with the presence of target site insensitivity due to kdr mutation and to increased metabolism through enzymatic activity. Kdr was expressed in both M and S forms. However, less than 1% of An. gambiae or Cx. quiqnuefasciatus showed the presence of the Ace.1(R) mutation. Carbamate/OP resistance was present at higher frequency in Culex than in An. gambiae. Dieldrin resistance was present in both species at all four localities. A higher frequency of pyrethroid-resistance was found in An. gambiae mosquitoes collected in urban areas compared to those collected in rice growing areas. The expansion of vegetable growing within urban areas probably contributed to selection pressure on mosquitoes. The detection of multiple resistance mechanisms in both An. gambiae and Cx. quinquefasciatus in Benin may represent a threat for the efficacy of ITNs and other forms of vector control such as indoor residual spraying in the future.
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              Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods

              Background Knockdown resistance (kdr) is a well-characterized mechanism of resistance to pyrethroid insecticides in many insect species and is caused by point mutations of the pyrethroid target site the para-type sodium channel. The presence of kdr mutations in Anopheles gambiae, the most important malaria vector in Africa, has been monitored using a variety of molecular techniques. However, there are few reports comparing the performance of these different assays. In this study, two new high-throughput assays were developed and compared with four established techniques. Methods Fluorescence-based assays based on 1) TaqMan probes and 2) high resolution melt (HRM) analysis were developed to detect kdr alleles in An. gambiae. Four previously reported techniques for kdr detection, Allele Specific Polymerase Chain Reaction (AS-PCR), Heated Oligonucleotide Ligation Assay (HOLA), Sequence Specific Oligonucleotide Probe – Enzyme-Linked ImmunoSorbent Assay (SSOP-ELISA) and PCR-Dot Blot were also optimized. The sensitivity and specificity of all six assays was then compared in a blind genotyping trial of 96 single insect samples that included a variety of kdr genotypes and African Anopheline species. The relative merits of each assay was assessed based on the performance in the genotyping trial, the length/difficulty of each protocol, cost (both capital outlay and consumable cost), and safety (requirement for hazardous chemicals). Results The real-time TaqMan assay was both the most sensitive (with the lowest number of failed reactions) and the most specific (with the lowest number of incorrect scores). Adapting the TaqMan assay to use a PCR machine and endpoint measurement with a fluorimeter showed a slight reduction in sensitivity and specificity. HRM initially gave promising results but was more sensitive to both DNA quality and quantity and consequently showed a higher rate of failure and incorrect scores. The sensitivity and specificity of AS-PCR, SSOP-ELISA, PCR Dot Blot and HOLA was fairly similar with a small number of failures and incorrect scores. Conclusion The results of blind genotyping trials of each assay indicate that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method. However, the cost of this assay, particularly in terms of initial capital outlay, is higher than that of some of the other methods. TaqMan assays using a PCR machine and fluorimeter are nearly as sensitive as real-time assays and provide a cost saving in capital expenditure. If price is a primary factor in assay choice then the AS-PCR, SSOP-ELISA, and HOLA are all reasonable alternatives with the SSOP-ELISA approach having the highest throughput.
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                Author and article information

                Journal
                Malar J
                Malaria Journal
                BioMed Central
                1475-2875
                2011
                8 June 2011
                : 10
                : 154
                Affiliations
                [1 ]Laboratoire de Recherche sur le Paludisme, Organisation de Coordination pour la lutte Contre les Endémies en Afrique Centrale (OCEAC), P.O. Box 288, Yaoundé, Cameroon
                [2 ]Faculty of Sciences, University of Yaoundé I, P.O. Box 337, Yaoundé, Cameroon
                [3 ]Institut de Recherche pour le Développement (IRD), UR 016, 911, avenue Agropolis, P.O. Box 64501, 34394 Montpellier cedex 5, France
                [4 ]Vector group, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, UK
                Article
                1475-2875-10-154
                10.1186/1475-2875-10-154
                3118161
                21651761
                ca729a7e-75bf-4fa1-b875-350e564386fc
                Copyright ©2011 Antonio-Nkondjio et al; licensee BioMed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 8 March 2011
                : 8 June 2011
                Categories
                Research

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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