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Background  Appropriate 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.    Methods  Here, 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.    Results  The 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.    Conclusion  The 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 .

malaria journal

Detection of the East and West African kdr mutation in Anopheles gambiae and Anopheles arabiensis from Uganda using a new assay based on FRET/Melt Curve analysis