Scalable and cost-effective methods for xenomonitoring of P. falciparum and antimalarial drug resistance validated with laboratory and wild-caught mosquitoes

Human blood samples serve as the gold standard for molecular surveillance of the malaria parasite Plasmodium falciparum. However, these samples may not accurately reflect the parasite population and come with logistical constraints and ethical requirements. Using blood-fed mosquitoes as a sample basis could overcome these challenges. We developed and validated DNA extraction methods and PCR assays, allowing for P. falciparum detection from whole mosquitoes and sequencing of drug resistance genes. PCRs were consistently positive on mosquito samples mimicking field conditions, i.e., with low P. falciparum infection (2000× dilution of a single oocyst-infected mosquito) or after a blood-meal with low parasitemia (250× dilution of 1% parasite density). Antimalarial drug resistance genes PfK13 and PfMDR1 could be sequenced from these samples, and markers of resistance could be detected in samples reflecting a mutated minority parasite population of ≥ 25%. Pools of 20 mosquitoes allowed for the detection of a single infected mosquito. We applied our protocol on 50 field-caught Anopheles mosquitoes from DR Congo. Five out of eight mosquito pools were P. falciparum positive, providing good sequencing reads for PfK13 and PfMDR1. Our presented method has the potential to facilitate surveillance, e.g., on drug resistance, leveraging the promising properties of xenomonitoring.