When a discriminating dose assay is not enough: measuring the intensity of insecticide resistance in malaria vectors

Abstract

Background: Guidelines from the World Health Organization for monitoring insecticide resistance in disease vectors recommend exposing insects to a predetermined discriminating dose of insecticide and recording the percentage mortality in the population. This standardized methodology has been widely adopted for malaria vectors and has provided valuable data on the spread and prevalence of resistance. However, understanding the potential impact of this resistance on malaria control requires a more quantitative measure of the strength or intensity of this resistance.

Methods: Bioassays were adapted to quantify the level of resistance to permethrin in laboratory colonies and field populations of Anopheles gambiae sensu lato. WHO susceptibility tube assays were used to produce data on mortality versus exposure time and CDC bottle bioassays were used to generate dose response data sets. A modified version of the CDC bottle bioassay, known as the Resistance Intensity Rapid Diagnostic Test (I-RDT), was also used to measure the knockdown and mortality after exposure to different multipliers of the diagnostic dose. Finally cone bioassays were used to assess mortality after exposure to insecticide treated nets.

Results: The time response assays were simple to perform but not suitable for highly resistant populations. After initial problems with stability of insecticide and bottle washing were resolved, the CDC bottle bioassay provided a reproducible, quantitative measure of resistance but there were challenges performing this under field conditions. The I-RDT was simple to perform and interpret although the end point selected (immediate knockdown versus 24 h mortality) could dramatically affect the interpretation of the data. The utility of the cone bioassays was dependent on net type and thus appropriate controls are needed to interpret the operational significance of these data sets.

Conclusions: Incorporating quantitative measures of resistance strength, and utilizing bioassays with field doses of insecticides, will help interpret the possible impact of resistance on vector control activities. Each method tested had different benefits and challenges and agreement on a common methodology would be beneficial so that data are generated in a standardized format. This type of quantitative data are an important prerequisite to linking resistance strength to epidemiological outcomes.

Fig. 1

Discriminating dose. Mortality rates after exposure to the WHO discriminating dose of permethrin. Error bars represent 95 % binomial confidence intervals. Columns with a different letter are significantly different to each other. The 90 % threshold used by WHO to define a resistant population is shown by a horizontal line

Fig. 2

Time response. Time response curves for adult females exposed to 0.75 % permethrin in WHO susceptibility tube assays. Best fit lines are plotted using XLSTAT. Tiefora and Tororo were exposed for five or six different time points ranging from 5 to 300 min. Tiassalé was exposed four time points from 30 min to 20 h

Fig. 3

Dose response. Dose response curves for adult females exposed to permethrin for 60 min in CDC bottle bioassays. Best fit lines are plotted using XLSTAT. A minimum of six concentrations were used for each strain

Fig. 4

CDC resistance intensity rapid diagnostic test. Knockdown after a 30 min exposure (open box), and mortality after a 24 h recovery (solid box) to bottles coated with different multitudes of the CDC bottle bioassay diagnostic dose for permethrin. Error bars represent 95 % binomial confidence intervals. The 90 % threshold used by CDC to define a resistant population is shown by a horizontal line

Fig. 5

Cone bioassays. Knockdown after a 3-min exposure (open box), and mortality after a 24-h recovery (solid box) to long-lasting insecticide-treated bed nets using a cone bioassay. Error bars represent 95 % binomial confidence intervals. The solid line and the dotted line represents the 80 % mortality and 95 % knockdown threshold used by WHO to test the efficacy of LLINs in phase I studies