Comparison of the standard WHO susceptibility tests and the CDC bottle bioassay for the determination of insecticide susceptibility in malaria vectors and their correlation with biochemical and molecular biology assays in Benin, West Africa

Abstract

Background: The detection of insecticide resistance in natural populations of Anopheles vectors is absolutely necessary for malaria control. In the African region, the WHO insecticide susceptibility test is the most common method for assessing resistance status. In order to search for a simple, rapid and more reliable technique in the assessment of insecticide resistance in malaria vectors, we compared the WHO tests with the CDC bottle bioassay in the Ouemé province of southern Benin where insecticide resistance has been widely reported.

Methods: Larvae and pupae of Anopheles gambiae s.l. mosquitoes were collected from the breeding sites in Ouemé. WHO and CDC susceptibility tests were conducted simultaneously on unfed female mosquitoes aged 2-5 days old. WHO bioassays were performed with impregnated papers of deltamethrin (0.05%) and bendiocarb (0.1%), whereas CDC bioassays were performed with stock solutions of deltamethrin (12.5 μg per bottle) and bendiocarb (12.5 μg per bottle). PCR techniques were used to detect species, Kdr and Ace-1 mutations. CDC biochemical assays using synergists were also conducted to assess the metabolic resistance.

Results: A slight decrease in mortality rates was observed with 97.95% and 98.33% obtained from CDC and WHO bioassays respectively in populations of mosquitoes from Adjara and Dangbo. PCR revealed that all specimens tested were Anopheles gambiae s.s. The Kdr mutation was found at high frequency in all populations and both the Kdr mutation and mono-oxygenase enzymes were implicated as mechanisms of pyrethroid resistance in An. gambiae from Misserete.

Conclusion: This study emphasizes that both WHO and CDC bioassays give similar results with regards to the susceptibility of mosquitoes to insecticides in southern Benin. There were complementarities between both methods, however, some specificity was noted for each of the two methods used. Both Kdr and metabolic mechanisms were implicated in the resistance.

Figure 1

Map of the study area.

Figure 2

Diagram for performing the CDC bottle bioassay with synergists[13].

Figure 3

Mortality of An. gambiae Kisumu, Adjara, Dangbo, Misserete and Seme populations observed after two hours exposure to CDC bottles treated with deltamethrin (1.25%).

Figure 4

Mortality of An. gambiae Kisumu, Adjara, Dangbo, Misserete and Seme populations observed after two hours exposure to CDC bottles treated with bendiocarb (1.25%).

Figure 5

Implication of mono-oxygenases in resistance of An. gambiae to pyrethroids in Misserete district.