Next-Generation Sequencing and Bioinformatics Protocol for Malaria Drug Resistance Marker Surveillance

Area of Science:

• Genomics and Molecular Biology
• Infectious Disease Surveillance
• Parasitology

Background:

• Tracking drug-resistant malaria parasites is crucial for effective disease control.
• Next-generation sequencing (NGS) offers advanced capabilities for molecular surveillance.
• Existing methods may not provide comprehensive data on multiple drug resistance genes simultaneously.

Purpose of the Study:

• To develop and validate an end-to-end NGS pipeline for molecular surveillance of P. falciparum drug resistance.
• To enable simultaneous tracking of allele frequencies for key drug resistance genes.
• To support routine surveillance of imported malaria cases and aid global adoption.

Main Methods:

• Development of the malaria resistance surveillance (MaRS) pipeline, integrating targeted amplicon deep sequencing (TADS) and bioinformatics.
• TADS involves PCR enrichment of specific P. falciparum genes (crt, mdr1, k13, dhfr, dhps, cytochrome b) and mitochondrial genome.
• Validation using 245 imported malaria cases, analyzing genotypes and single nucleotide polymorphisms at the individual patient level.

Main Results:

• High prevalence of resistance markers: 42% crt CVIET (chloroquine resistance), 92% dhpsIRN (pyrimethamine resistance), 26% dhps SGEAA (sulfadoxine resistance), 40% mdr1 NFSND.
• Low prevalence of artemisinin resistance (k13 alleles) and high susceptibility (99%) to atovaquone-proguanil among imported cases.
• The MaRS pipeline effectively captured allele frequencies for multiple drug resistance genes.

Conclusions:

• The MaRS pipeline provides an effective tool for molecular surveillance of malaria drug resistance.
• Implementation at the CDC and global partners can enhance imported malaria case management and control strategies.
• NGS-based surveillance is vital for monitoring evolving drug resistance patterns in P. falciparum.