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

Abstract

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 Plasmodium parasites and Anopheles mosquito vectors are key components of effective malaria control decision-making. Previously, we have designed and implemented a nanopore-based workflow for targeted P. falciparum 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 merozoite surface protein 2 ( msp2), and the 18S ribosomal RNA (rRNA) gene for Plasmodium 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. Malaria is a major cause of disease and death globally, especially for young children living in Africa. Affected countries aim to reduce the spread of the malaria parasite, which is transmitted by mosquitoes, and to treat infected people using drugs. However, the parasites can develop resistance to the drugs, making them less effective. Newly approved malaria vaccines are also being deployed at scale. It is therefore important to monitor parasites for the development and spread of drug resistance and for any changes relevant to the vaccine, to ensure treatment outcomes remain optimal. This study describes a laboratory method for monitoring malaria parasites suitable for use in tropical areas where malaria is common. Starting from a blood sample taken from an infected person, targeted sections of malaria parasite DNA are amplified and sequenced using nanopore technology. The genes analysed provide information on drug resistance, the current vaccine target, and which malaria species are present. The study also describes laboratory practices to ensure high quality standards are maintained consistently. In summary, this study reports a method for monitoring malaria parasites using a streamlined DNA sequencing workflow; further work can assess its performance in malaria zones and develop protocols suitable for clinical use.