Assessing <italic>Plasmodium falciparum</italic> transmission in mosquito-feeding assays using quantitative PCR.

Background: Evaluating the efficacy of transmission-blocking interventions relies on mosquito-feeding assays, with transmission typically assessed by microscopic identification of oocysts in mosquito midguts; however, microscopy has limited throughput, sensitivity and specificity. Where low prevalence and intensity mosquito infections occur, as observed during controlled human malaria infection studies or natural transmission, a reliable method for detection and quantification of low-level midgut infection is required. Here, a semi-automated, Taqman quantitative PCR (qPCR) assay sufficiently sensitive to detect a single-oocyst midgut infection is described. Results: Extraction of genomic DNA from <italic>Anopheles stephensi</italic> midguts using a semi-automated extraction process was shown to have equivalent extraction efficiency to manual DNA extraction. An 18S <italic>Plasmodium falciparum</italic> qPCR assay was adapted for quantitative detection of <italic>P. falciparum</italic> midgut oocyst infection using synthetic DNA standards. The assay was validated for sensitivity and specificity, and the limit of detection was 0.7 genomes/µL (95% CI 0.4–1.6 genomes/µL). All microscopy-confirmed oocyst infected midgut samples were detected by qPCR, including all single-oocyst positive midguts. The genome number per oocyst was assessed 8–9 days after feeding assay using both qPCR and droplet digital PCR and was 3722 (IQR: 2951–5453) and 3490 (IQR: 2720–4182), respectively. Conclusions: This semi-automated qPCR method enables accurate detection of low-level <italic>P. falciparum</italic> oocyst infections in mosquito midguts, and may improve the sensitivity, specificity and throughput of assays used to evaluate candidate transmission-blocking interventions. [ABSTRACT FROM AUTHOR]