Detection of knockdown resistance (kdr) mutations in Anopheles gambiae: a comparison of two new high-throughput assays with existing methods
Chris Bass,Dimitra Nikou,Martin J. Donnelly,Martin S. Williamson,Hilary Ranson,Amanda Ball,John Vontas,Linda M. Field +7 more
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Two new high-throughput assays were developed and compared with four established techniques for kdr detection, indicating that where maximum sensitivity and specificity are required the TaqMan real-time assay is the preferred method.Abstract:
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. 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). 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. 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.read more
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Effectiveness of a long-lasting piperonyl butoxide-treated insecticidal net and indoor residual spray interventions, separately and together, against malaria transmitted by pyrethroid-resistant mosquitoes: a cluster, randomised controlled, two-by-two factorial design trial.
Natacha Protopopoff,Jacklin F. Mosha,Eliud Lukole,J. D. Charlwood,Alexandra Wright,Charles D Mwalimu,Alphaxard Manjurano,Franklin W. Mosha,William Kisinza,Immo Kleinschmidt,Immo Kleinschmidt,Mark Rowland +11 more
TL;DR: The PBO long- lasting insecticidal net and non-pyrethroid indoor residual spraying interventions showed improved control of malaria transmission compared with standard long-lasting insecticidal nets where pyrethroid resistance is prevalent and either intervention could be deployed to good effect.
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Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana
Sara N. Mitchell,Bradley J. Stevenson,Pie Müller,Craig S. Wilding,Alexander Egyir-Yawson,Stuart G. Field,Janet Hemingway,Mark J. I. Paine,Hilary Ranson,Martin J. Donnelly +9 more
TL;DR: In the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide, which is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of Insecticide routinely used for malaria control.
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Multiple-Insecticide Resistance in Anopheles gambiae Mosquitoes, Southern Côte d’Ivoire
TL;DR: Development of newer classes of insecticide is crucial because if resistance continues unchecked, the hard-earned progress in malaria control in Africa could be quickly reversed.
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Lessons from the past: managing insecticide resistance in malaria control and eradication programmes
TL;DR: This work outlines the key requirements for establishing an insecticide resistance surveillance system and urges all those involved in malaria vector control, either directly or as facilitators, to ensure that these measures are incorporated into control programmes.
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Footprints of positive selection associated with a mutation (N1575Y) in the voltage-gated sodium channel of Anopheles gambiae
Christopher M. Jones,Milindu Liyanapathirana,Fiacre R. Agossa,David Weetman,Hilary Ranson,Martin J. Donnelly,Craig S. Wilding +6 more
TL;DR: This work documents the emergence of a mutation, N1575Y, within the linker between domains III-IV of the VGSC, suggesting that the N15 75Y mutation compensates for deleterious fitness effects of 1014F and/or confers additional resistance to insecticides.
References
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Journal ArticleDOI
Identification of Single Specimens of the Anopheles Gambiae Complex by the Polymerase Chain Reaction
TL;DR: A ribosomal DNA-polymerase chain reaction (PCR) method has been developed for species identification of individuals of the five most widespread members of the Anopheles gambiae complex, a group of morphologically indistinguishable sibling mosquito species that includes the major vectors of malaria in Africa.
Journal ArticleDOI
Allelic discrimination using fluorogenic probes and the 5' nuclease assay.
TL;DR: The 5' nuclease (TaqMan) as discussed by the authors is a typical PCR that uses a fluorogenic probe, consisting of an oligonucleotide labeled with both a fluorescent reporter dye and a quencher dye.
Journal ArticleDOI
Molecular characterization of pyrethroid knockdown resistance (kdr) in the major malaria vector Anopheles gambiae s. s.
David Martínez-Torres,Fabrice Chandre,Martin S. Williamson,Frédéric Darriet,Jean Baptiste Berge,Alan L. Devonshire,Pierre Guillet,Nicole Pasteur,David Pauron +8 more
TL;DR: This work demonstrates that a modification of the voltage‐gated sodium channel protein recently shown to be associated with mutations of the para‐type sodium channel gene is present in certain strains of pyrethroid resistant A. gambiae, and describes a PCR‐based diagnostic test allowing its detection in the genome of single mosquitoes.
Journal ArticleDOI
Genotyping of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small Amplicons
Michael Liew,Robert J. Pryor,Robert Palais,Cindy Meadows,Maria Erali,Elaine Lyon,Elaine Lyon,Carl T. Wittwer,Carl T. Wittwer +8 more
TL;DR: In this article, high-resolution melting of PCR amplicons with the DNA dye LCGreen™ I was recently introduced as a homogeneous, closed-tube method of genotyping that does not require probes or real-time PCR.
Journal ArticleDOI
Identification of a point mutation in the voltage-gated sodium channel gene of Kenyan Anopheles gambiae associated with resistance to DDT and pyrethroids.
TL;DR: The diagnostic PCR was adapted to detect the leucine–serine mutation and it was demonstrated that this kdr allele was present in individuals collected from the Kenyan trial site in 1986, prior to the introduction of pyrethroid‐impregnated bednets.