# Multiplex long-amplicon sequencing for comprehensive molecular surveillance of Plasmodium falciparum resistance to artemisinin and partner drugs in artemisinin-based combination therapies (ACTs)

## TL;DR

This study develops a new sequencing method to detect drug resistance in malaria parasites, enabling comprehensive and cost-effective monitoring of resistance mutations.

## Contribution

The study introduces a multiplex long-amplicon sequencing panel for full-gene surveillance of Plasmodium falciparum drug resistance.

## Key findings

- The panel covers six genes, including four artemisinin resistance markers and two ACT partner drug resistance markers.
- It achieves high sensitivity and species-specific amplification with low sequencing data requirements and minimal cost per sample.
- The method enables detection of both known and emerging resistance mutations across entire genes.

## Abstract

Antimalarial resistance, especially artemisinin resistance, in Plasmodium falciparum threatens global malaria control. Molecular surveillance of Plasmodium falciparum drug resistance is critical for monitoring the efficacy of artemisinin-based combination therapies (ACTs). Current molecular surveillance tools for Plasmodium falciparum artemisinin resistance are restricted to predefined polymorphism hotspots in limited loci (Pfk13 domain), failing to capture novel mutations in emerging resistance genes (Pfcoronin, Pfubp1, Pfap2μ) or cover complete coding regions. This study addresses this gap by developing an optimized long-amplicon panel for comprehensive, full-gene resistance surveillance.

Six genes were selected for the multiplex PCR amplification, including four artemisinin resistance-related markers (Pfk13, Pfcoronin, Pfap2μ and Pfubp1) and two ACTs partner drugs resistance markers (Pfmdr1 and Pfcrt). Amplicons were standardized to 2.5 ± 0.2 kb using multiply software to minimize amplification bias. The full-length coverage of Pfk13, Pfcoronin and Pfap2μ was achieved. Primer concentrations and annealing temperatures were iteratively optimized through gel electrophoresis and sequencing validation. Analytical validation was assessed using 11 mock samples (parasitemia ranging from 1% to 0.0001%) and 16 field-collected venous blood (VB) samples. The samples were processed via Illumina paired-end sequencing.

The long-amplicon panel was specifically designed to cover the full-length coding region of the Pfk13 gene and co-amplify three artemisinin resistance-related molecular markers. It can exhibited species-specific amplification efficiency for Plasmodium falciparum targets with undetectable cross-reactivity against non-falciparum Plasmodium species. Analytical sensitivity thresholds were at 50 parasites/μL for dried blood spots (DBS) sample and 5 parasites/μL for VB sample, with all targets achieving 100% coverage. Notably, DBS samples over 50 p/μL required only 0.25GB of sequencing data (mean depth: 55 ×) for complete target coverage, while VB samples above 5 parasites /μL required 0.5GB data (mean depth: 33 ×) still keeping over 89% coverage uniformity. The total cost per sample was minimized to $15.60, encompassing PCR amplification, library preparation and sequencing.

The new long-amplicon panel improves malaria molecular drug-resistance monitoring by detecting known and emerging mutations across entire genes in a single test. The high sensitivity for ultralow parasitemia samples, coupled with their species specificity and cost-effectiveness, makes it a scalable tool for monitoring multidrug-resistant P. falciparum strains, particularly in resource-limited settings.

The online version contains supplementary material available at 10.1186/s13071-025-07058-6.