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

ABSTRACT

Background

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.

Research design and methods

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.

Results

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.

Conclusions

We developed highly sensitive and efficient (RT-) ddPCR assays for contemporary operational needs that require monitoring of low malaria transmission and emerging insecticide resistance.

KEYWORDS:

• ddpcr
• infectious diseases
• insecticide resistance
• malaria
• Plasmodium falciparum
• vector monitoring

Supplemental Material

Supplemental data for this article can be accessed here

Acknowledgments

The following reagent was obtained through BEI Resources, NIAID, NIH: Anopheles gambiae, Anopheles gambiae KISUMU1 Bulk Frozen, MRA-762B. We thank the Liverpool Insect Testing Establishment (LITE) for providing the VK7 Anopheles gambiae laboratory strain, as well as theTropIQ Health Sciences and Radboud University Medical Center for providing the P. falciparum infected mosquito samples. We thank Prof. Sofia Agelaki and the Laboratory of Translational Oncology at the School of Medicine of the University of Crete, for kindly providing us access to the ddPCR platform.

This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project “Reinforcement of Postdoctoral Researchers - 2nd Cycle” (MIS-5033021), implemented by the State Scholarships Foundation (ΙΚΥ). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 731060 (INFRAVEC2).

Reviewer Disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Author Contributions

K Mavridis and J Vontas were involved in the conception and design of the study. K Mavridis and K Michaelidou performed the experiments, analysis and interpretation of the data. All authors were involved in drafting the paper and gave the final approval to be published. All authors agree to be accountable for all aspects of the work.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Additional information

Funding

This research is co-financed by Greece and the European Union (European Social Fund- ESF) through the Operational Programme «Human Resources Development, Education and Lifelong Learning» in the context of the project ‘Reinforcement of Postdoctoral Researchers - 2nd Cycle’ (MIS-5033021), implemented by the State Scholarships Foundation (ΙΚΥ). This project has received funding from the European Union’s Horizon 2020 research and innovation program under grant agreement No 731060 (INFRAVEC2).