Most of the specimens collected in Pointe-Noire were
An. gambiae S-form (98.1%). Unexpectedly, one specimen was identified as a hybrid M/S whilst no
An. gambiae M-form were collected in this study area. Koekemoer
et al (Multiple insecticide resistance in
Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo, submitted) published additional data from this area from 343 specimens. This was the only hybrid identified from large sample analysis and additional genetic studies would need to be concluded to explain the presence of the hybrid in the rare presence of
An. gambiae M-form (0.3%) by performing analysis of large sample size. The presence of the
kdr mutations within this study cohort was unexpectedly high (100% of sequenced samples; 96% of hydrolysis probe analysis samples). Both
kdr-e and
kdr-w resistance mutations occurred simultaneously in 29 (55.8%) out of 52 specimens. The frequencies of the homozygous
kdr-e and
kdr-w single mutations were 7 (13.5%) and 14 specimens (26.9%) respectively. There was one specimen heterozygous for the
kdr-e (1.9%) and another heterozygous for the
kdr-w (1.9%) mutations. Although recent studies reported that the frequencies of the both mutations present at the same time were much lower (1.7-16.1% in Cameroon [
6‐
8] and 1.4% in Uganda [
10]) than the present study, Pinto
et al [
9] reported that all
An. gambiae specimens collected in Gabon in 2000 carried the
kdr-e or
kdr-w mutations and 55.7% of the co-occurring both mutations. A recent study has reported that the
An. gambiae population from Congo was resistant to DDT (100%), deltamethrin (26%), dieldrin (69%) and bendiocarb (3%) (Koekemoer
et al: Multiple insecticide resistance in
Anopheles gambiae (Diptera: Culicidae) from Pointe Noire, Republic of the Congo, submitted). This study also suggested that both
kdr mutations in
An. gambiae are widespread in the region.
Although DNA sequencing remains the most accurate method for detecting the presence of
kdr mutations, it is much more time consuming and expensive (> US$10 per sample for sequencing in both directions) than other assays. Bass
et al [
27] reported that the hydrolysis probe analysis is the most sensitive and specific assay for detecting
kdr mutations (5.2% of failed reactions and 0% of mis-scores) when compared to AS-PCR, HRM, HOLA, SSOP-ELISA and PCR-Dot Blot assays even though the cost of the assay (US$1.72 per sample) is higher than some of the other methods (US$0.62 - US$ 1.74 per sample). In this study, 4% (2/52) samples gave different results between the hydrolysis probe and sequencing assays due to a failed reaction of either the
kdr-e or
kdr-w PCR.
Results from this study recommend that the allelic discrimination axes used for detecting both mutations simultaneously can be adjusted to increase sensitivity of the assay. This is especially true in those populations carrying both mutations at high frequency. However, this decision should be made cautiously and sequencing should be used to validate the adjustments made.
The accuracy of the assay could fluctuate due to the determination of the allelic discrimination values for the axes as well as due to other variable running conditions. A number of conditions such as quality and quantity of DNA templates, master mixes, PCR assay performers, the positioning of the allelic discrimination axes and other conditions might affect kdr genotyping. The best method for genotyping seems to be end point analysis as well as intensity of fluorescence, although two out of fifty two samples in this study could not be genotyped using these values due to misleading RFU values, even after adjustment of the default allelic discrimination axes.