Ley, B. | Search bioRxiv

bioRxiv2025

Rapid detection of G6PD deficiency SNPs using a novel amplicon-based MinION Sequencing Assay

Plasmodium vivax malaria remains a significant global health challenge, complicated by the parasites ability to form dormant liver stages (hypnozoites) that cause relapses. Radical cure of P. vivax malaria requires administration of a hypnozoitocidal drug, such as primaquine or tafenoquine. However, these drugs can cause severe haemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. G6PD deficiency is caused by more than 230 different variants at the gene level that confer different degrees of deficiency phenotypically. Understanding the distribution of different G6PD variants in affected populations is essential to inform safer antimalarial treatment strategies. This study aimed to develop a cost-effective sequencing assay targeting key regions of the G6PD gene, suitable for field deployment. A novel assay based on Nanopore technology was designed to amplify two amplicons covering exon 3 to exon 13, focusing on known variants associated with enzyme deficiency. A total of 79 samples from individuals in Cambodia, Vietnam, Afghanistan, and China were sequenced, and a bioinformatics pipeline was created for the targeted variant calling of 192 G6PD SNP mutations. The assay demonstrated reliable detection of known variants, with high concordance between runs, within runs, and with Sanger sequencing. The Nanopore MinION long-amplicon sequencing assay offers a robust and portable solution for large-scale G6PD genotyping in low-resource settings, that will improve malaria control and elimination strategies by enabling safer antimalarial treatment.

microbiology↗

bioRxiv2019

A molecular barcode and online tool to identify and map imported infection with Plasmodium vivax

Imported cases present a considerable challenge to the elimination of malaria. Traditionally, patient travel history has been used to identify imported cases, but the long-latency liver stages confound this approach in Plasmodium vivax. Molecular tools to identify and map imported cases offer a more robust approach, that can be combined with drug resistance and other surveillance markers in high-throughput, population-based genotyping frameworks. Using a machine learning approach incorporating hierarchical FST (HFST) and decision tree (DT) analysis applied to 831 P. vivax genomes from 20 countries, we identified a 28-Single Nucleotide Polymorphism (SNP) barcode with high capacity to predict the country of origin. The Matthews correlation coefficient (MCC), which provides a measure of the quality of the classifications, ranging from -1 (total disagreement) to 1 (perfect prediction), exceeded 0.9 in 15 countries in cross-validation evaluations. When combined with an existing 37-SNP P. vivax barcode, the 65-SNP panel exhibits MCC scores exceeding 0.9 in 17 countries with up to 30% missing data. As a secondary objective, several genes were identified with moderate MCC scores (median MCC range from 0.54-0.68), amenable as markers for rapid testing using low-throughput genotyping approaches. A likelihood-based classifier framework was established, that supports analysis of missing data and polyclonal infections. To facilitate investigator-lead analyses, the likelihood framework is provided as a web-based, open-access platform (vivaxGEN-geo) to support the analysis and interpretation of data produced either at the 28-SNP core or full 65-SNP barcode. These tools can be used by malaria control programs to identify the main reservoirs of infection so that resources can be focused to where they are needed most.

molecular biology↗