An expanded method for malaria parasite genetic surveillance using targeted nanopore sequencing.

Malaria causes around 250 million cases and over 600,000 deaths annually, with the heaviest burden falling on young children living in sub-Saharan Africa. Molecular surveillance of parasites and mosquito vectors are key components of effective malaria control decision-making. Previously, we have designed and implemented a nanopore-based workflow for targeted molecular surveillance in Ghana, which we call DRAG1 (drug resistance + antigen multiplex PCR). Here, we describe an updated and expanded multiplex assay ('DRAG2') with additional amplicon targets that incorporate more antimalarial drug resistance markers, the polymorphic surface antigen ( ), and the 18S ribosomal RNA (rRNA) gene for species detection. We describe the performance of the DRAG2 assay over a range of parasitaemias and sample types (venous blood and dried blood spots), with suggested systems of quality control including the use of synthetic plasmids for positive controls and recommended coverage thresholds. The plasmids are highly economical, and engineered to include both 'test' single nucleotide polymorphisms (SNPs), such as known drug resistance markers, and 'control' SNPs, which are not found in nature and thus signal contamination if detected in clinical samples. We provide standard operating procedures (SOPs) for use by teams aiming to implement the assay in their laboratory. In summary, we describe an updated nanopore-based method for malaria molecular surveillance, including detailed consideration of quality control processes and SOPs. These are important steps in the transition from research tool to diagnostic assay, which will require further testing in endemic settings and regulatory processes and approvals.