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Background  Knockdown resistance ( kdr ) caused by a single base pair mutation in the sodium channel gene is strongly associated with pyrethroid insecticide resistance in  Anopheles gambiae  in West-Central Africa. Recently, various molecular techniques have been developed to screen for the presence of the  kdr  mutations in vector populations with varying levels of accuracy. In this study, the results of the hydrolysis probe analysis for detecting the  kdr  mutations in  An. gambiae  s.s. from the Republic of the Congo were compared with DNA sequence analysis.    Methods  A total of 52 pyrethroid and DDT resistant  An. gambiae  from Pointe-Noire (Congo-Brazzaville) were tested for detection of the two  kdr  mutations ( kdr -e and  kdr -w) that are known to occur in this species. Results from the hydrolysis probe analysis were compared to DNA sequencing to verify the accuracy of the probe analysis for this vector population.    Results  Fifty-one specimens were found to be  An. gambiae  S-form and one was a M/S hybrid. DNA sequencing revealed that more than half of the specimens (55.8%) carried both the  kdr -e and  kdr -w resistance mutations, seven specimens (13.5%) were homozygous for the  kdr -e mutation, and 14 specimens (26.9%) were homozygous for the  kdr -w mutation. A single individual was genotyped as heterozygous  kdr -e mutation (1.9%) only and another as heterozygous  kdr -w mutation (1.9%) only. Analysis using hydrolysis probe analysis, without adjustment of the allelic discrimination axes on the scatter plots, revealed six specimens (11.5%) carrying both mutations, 30 specimens (57.8%) as homozygous  kdr -w, six specimens (11.5%) homozygous for the  kdr -e mutation, one specimen (1.9%) heterozygous for the  kdr -w mutation and one specimen (1.9%) present in wild type form. Eight of the specimens (15.4%) could not be identified using unadjusted hydrolysis probe analysis values. No heterozygous  kdr -e mutations were scored when adjustment for the allelic discrimination axes was omitted. However, when the axes on the scatter plots were adjusted the results were consistent with those of the DNA sequence analysis, barring two individuals that were mis-scored in the hydrolysis probe analysis.    Conclusion  Both the  kdr -e and  kdr -w mutations were abundant in  An. gambiae  S-form from Pointe-Noire. The hydrolysis probe analysis can lead to misleading results if adjustment to allelic discrimination axes is not investigated. This is mainly relevant when both  kdr -e and  kdr -w are present in a population in a high frequency. This report highlights the importance of concurrent screening for both mutations. Therefore, performing routine assay protocols blindly can result in the misinterpretation of results. Although hydrolysis probe analysis of  kdr  is still held as the gold standard assay, this paper highlights the importance of  kdr  mutation confirmation via sequencing especially in regions where  kdr  frequency has never been reported before or where both the  kdr -e and  kdr -w mutations are present simultaneously.

malaria journal

A comparison of DNA sequencing and the hydrolysis probe analysis (TaqMan assay) for knockdown resistance (kdr) mutations in Anopheles gambiae from the Republic of the Congo