Abstract
BackgroundAppropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. The malaria vectors Anopheles gambiae s.s. and Anopheles arabiensis have developed resistance to pyrethroid insecticides as a result of a mechanism conferring reduced nervous system sensitivity, better known as knockdown resistance (kdr). In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations. However, these AS-PCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results.MethodsHere, a new assay for the detection of knockdown resistance in An. gambiae s.s. and An. arabiensis based on Fluorescence Resonance Energy Transfer/Melt Curve analysis (FRET/MCA) is presented and compared with the existing assays.ResultsThe new FRET/MCA method has the important advantage of detecting both kdr alleles in one assay. Moreover, results show that the FRET/MCA is more reliable and more sensitive than the existing AS-PCR assays and is able to detect new genotypes. By using this technique, the presence of the East African kdr mutation (L1014S) is shown for the first time in An. arabiensis specimens from Uganda. In addition, a new kdr genotype is reported in An. gambiae s.s. from Uganda, where four An. gambiae s.s. mosquitoes possess both, the West (L1014F) and East (L1014S) African kdr allele, simultaneously.ConclusionThe presence of both kdr mutations in the same geographical region shows the necessity of a reliable assay that enables to detect both mutations in one single assay. Hence, this new assay based on FRET/MCA will improve the screening of the kdr frequencies in An. gambiae s.s. and An. arabiensis.
Highlights
Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes
The melt curve obtained for mosquitoes homozygous for the L1014F, the L1014S or the wild type allele was identical to the melt curve obtained for the corresponding plasmid (Figure 3B)
The FRET/MCA assay allows the genotyping of An. gambiae s.s. and An. arabiensis specimens for both kdr alleles by the use of only one probe
Summary
Appropriate monitoring of vector resistance to insecticides is an integral component of planning and evaluation of insecticide use in malaria control programmes. In An. gambiae s.s. and An. arabiensis, two different substitutions in the para-type sodium channel, a L1014F substitution common in West Africa and a L1014S replacement found in Kenya, are linked with kdr. Two different allele-specific polymerase chain reactions (AS-PCR) are needed to detect these known kdr mutations These ASPCR assays rely on a single nucleotide polymorphism mismatch, which can result in unreliable results. An important resistance mechanism against pyrethroids and DDT, known as knockdown resistance (kdr), has been linked to a single mutation in the para-type sodium channel gene in several insect species [2]. Two different kdr mutations have been found in the African malaria vector Anopheles gambiae s.s. In West Africa, knockdown resistance is linked with a mutation resulting in a leucine to phenylalanine substitution in the S6 segment of domain II of the para-type sodium channel (L1014F) [3]. The leucine to phenylalanine mutation was found in An. arabiensis samples from Burkina Faso [5] and the leucine to serine mutation was found in the same species from Kenya [6]
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