Abstract
Background
The use of insecticides for vector control is central to malaria control programs. Resistance to commonly used pyrethroids and DDT insecticides are widespread and threaten the success of these programs. Diagnostic markers for insecticide resistance (IR) are difficult to validate, but knock down resistance (kdr) in the major malarial vector An. gambiae is well characterised.
The early detection of these markers could better inform vector control programs. In remote field settings where gathering reliable bioassay data is challenging, it is important to have field-applicable diagnostic tools. Existing tests for the sensitive detection of kdr mutations are only suitable for use in well-equipped laboratory settings. There is an unmet need for field-applicable diagnostics.
Methods
A new diagnostic test was developed to target kdr mutations in An. gambiae mosquitoes. Three mutations in the VGSC gene were targeted due to their known interactive effects on kdr: L1014F, L1014S and N1575Y. The test was developed on the portable, rapid, point-of-care compatible PCR platform – the Genedrive® (Genedrive® plc), making it better suited for use in the field. Using either crude mosquito lysates or extracted DNA, the Genedrive analyses the melt curves of amplified DNA to differentiate between wild-type and mutant alleles.
Results
With an assay runtime of under an hour, discernible melt peaks were observed for each of the different genotypes. The test utilises a single GeneDrive cartridge for the detection of 3 kdr mutations. Preliminary validation of the test comprised a panel of 70 previously extracted DNA samples from field-caught An. gambiae mosquitoes. Our test showed complete agreement with the widely-used TaqMan reference test, achieving a sensitivity and specificity of 100%.
Conclusion
We developed a sensitive diagnostic test for use on the Genedrive® platform, as the first field-ready diagnostic for kdr.