Methods in Anopheles Research - MR4

Chapter 5 : Insecticide Resistance5.1 Insecticide Resistance Bioassays5.1.3 Guidelines for Evaluating Insecticide Resistance in Vectors using the CDC Bottle BioassayPage 21 of 24Appendix 2. Diagnostic doses and CDC bottle bioassay calibrationIt is assumed that resistance is present if a diagnostic dose, proven and validated against a susceptibleinsect population, is survived by members of a test population at a predetermined diagnostic time. Thediagnostic dose and diagnostic time are optimal parameters for detecting insecticide resistance. Adiagnostic dose that is too low will not kill susceptible mosquitoes during the bioassay, providing a falsepositiveresult for resistance. On the other hand, a diagnostic dose that is too high will kill resistantmosquitoes during the bioassay, masking resistance.For some insect vectors from some geographic regions, diagnostic doses and diagnostic times for severalinsecticides have already been determined. It is recommended that countries in these regions use thealready established parameters to allow them to compare data across countries or within regions.However, if this information is not available, the diagnostic dose and the diagnostic time will need to bedefined for each insecticide, in each region, and for each main vector species that is to be monitored. Todetermine the diagnostic dose and the diagnostic time for use in the CDC bottle bioassay, the assay willhave to be calibrated.Calibration assayThe assay is calibrated by first selecting the testing population and possible lengths of test time, and thenby determining possible diagnostic doses, given preferred diagnostic times.Population: The first step is to select a susceptible vector population to use as a baseline. If such apopulation is not available, it is possible to use the vector population from the area where the chemicalvector control measures are to be applied. This will be the reference point against which all futurepopulations can be compared.Diagnostic time: For practical reasons, the diagnostic time should be between 30 and 60 minutes.Diagnostic dose: The diagnostic dose will be a dose of insecticide that can kill 100% of mosquitoessometime between 30 and 60 minutes and that is below the saturation point. To determine possiblediagnostic doses, first prepare bottles with a range of different concentrations of insecticides per bottle, asoutlined in the guideline. Using each of these different bottles, run separate CDC bottle bioassays on 25mosquitoes of the susceptible population to determine upper limit of the diagnostic dose, which is thesaturation point. The saturation point can be defined as a concentration above which no additionaldecrease in the time required to kill 100% of the mosquitoes with an increase in insecticide concentration.See a more detailed explanation on how to determine the saturation point below.It may be necessary to run additional sets of concentrations around that range until the optimal diagnosticdose is determined. For example, starting with 10 µg/bottle, increase concentration with increments of 5µg/bottle, and continue to a final concentration of 200 µg/bottle. If no clear saturation point can bedetermined, run more assays using bottles with 200 µg/bottle, with increments of 5µg. If the saturation point still cannot be determined, more assays may be run with bottles using smallerincrements of insecticide.Interpretation of calibration dataGraphing the results of the calibration assay will show that the time-mortality line becomes straighter,steeper, and moves toward the Y-axis as the insecticide concentration increases (Figure 5.1.3.4). Thismeans that by increasing insecticide concentration the time-mortality line will reach a point whereincreasing the concentration of insecticide will not kill all mosquitoes any faster. In the example below, 15