Molecular malaria surveillance using a novel protocol for extraction and analysis of nucleic acids retained on used rapid diagnostic tests.

The use of malaria rapid diagnostic tests (RDTs) as a source for nucleic acids that can be analyzed via nucleic acid amplification techniques has several advantages, including minimal amounts of blood, sample collection, simplified storage and shipping conditions at room temperature. We have systematically developed and extensively evaluated a procedure to extract total nucleic acids from used malaria RDTs. The co-extraction of DNA and RNA molecules from small volumes of dried blood retained on the RDTs allows detection and quantification of P. falciparum parasites from asymptomatic patients with parasite densities as low as 1 Pf/µL blood using reverse transcription quantitative PCR. Based on the extraction protocol we have developed the ENAR (Extraction of Nucleic Acids from RDTs) approach; a complete workflow for large-scale molecular malaria surveillance. Using RDTs collected during a malaria indicator survey we demonstrated that ENAR provides a powerful tool to analyze nucleic acids from thousands of RDTs in a standardized and high-throughput manner. We found several, known and new, non-synonymous single nucleotide polymorphisms in the propeller region of the kelch 13 gene among isolates circulating on Bioko Island, Equatorial Guinea.