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

Area of Science:

• Molecular biology
• Parasitology
• Genomics

Background:

• Malaria remains a significant global health threat, causing millions of cases and hundreds of thousands of deaths annually, disproportionately affecting children in sub-Saharan Africa.
• Effective malaria control relies on molecular surveillance of Plasmodium parasites and Anopheles mosquito vectors.
• Previous work established a nanopore-based workflow (DRAG1) for P. falciparum surveillance in Ghana.

Purpose of the Study:

• To describe an updated and expanded multiplex assay, DRAG2, for enhanced malaria molecular surveillance.
• To incorporate additional antimalarial drug resistance markers, merozoite surface protein 2 (msp2), and 18S ribosomal RNA (rRNA) for Plasmodium species detection.
• To provide quality control measures and standard operating procedures (SOPs) for assay implementation.

Main Methods:

• Development and validation of the DRAG2 nanopore-based multiplex assay.
• Testing assay performance across various parasitaemias and sample types (venous blood, dried blood spots).
• Utilizing synthetic plasmids with engineered single nucleotide polymorphisms (SNPs) for quality control, including 'test' and 'control' SNPs.

Main Results:

• The DRAG2 assay successfully incorporates new targets for drug resistance and Plasmodium species identification.
• Performance was evaluated across different parasitaemias and sample types, demonstrating assay robustness.
• Economical synthetic plasmids were engineered for reliable positive controls and contamination detection.

Conclusions:

• The DRAG2 assay represents an advancement in nanopore-based malaria molecular surveillance.
• Detailed quality control procedures and SOPs are provided, facilitating the transition from research to diagnostic application.
• Further validation in endemic settings and regulatory approval are necessary for widespread diagnostic use.