Highly sensitive droplet digital PCR-based diagnostics for the surveillance of malaria vector populations in low transmission and incipient resistance settings

Sensitive monitoring of Plasmodium infective mosquitoes in low malaria transmission settings is of high priority for disease control. Early detection of insecticide resistance at low frequencies is also key for vector monitoring nowadays, when new insecticides are launched to control vector populations. An. gambiae mosquitoes with predetermined infection and resistance status were used to produce populations with various malaria infection rates and mutant allelic frequencies (MAFs) of target site insecticide resistance traits. Total RNA and gDNA were isolated and used in droplet Digital PCR (ddPCR) and Reverse Transcription (RT) ddPCR performed in the QX200 ddPCR System. We developed a novel ddPCR for detecting P. falciparum DNA in pooled mosquito head-thoraces with infective rate as low as 1.0%. A dissection-free RT-ddPCR assay for specific infective-stage detection was additionally developed and validated (accuracy = 100%) in mosquito pools with infective rates down to 1.0%. A novel ddPCR assay for insecticide resistant alleles, which was able to reliably quantify MAFs as low as 0.050% in pooled mosquito specimens, is also reported. We developed highly sensitive and efficient (RT-) ddPCR assays for contemporary operational needs that require monitoring of low malaria transmission and emerging insecticide resistance.