High-Throughput Ultrasensitive Molecular Techniques for Quantifying Low-Density Malaria Parasitemias

The epidemiology of malaria in “low-transmission ” areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (5l) and therefore cannot detect parasite densities of<200/ml. Their use underestimates true para-site carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive “high-volume ” quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of>250l and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PC