A next-generation sequencing and bioinformatics protocol for Malaria drug Resistance marker Surveillance (MaRS) [PublishAheadOfPrint]

The recent advances in next-generation sequencing technologies provide a new and effective way of tracking malaria drug resistant parasites. To take advantage of this technology an end-to-end Illumina targeted amplicon deep sequencing (TADS) and bioinformatics pipeline for molecular surveillance of drug resistance in P. falciparum, called Malaria Resistance Surveillance (MaRS), was developed. TADS relies on PCR enriching genomic regions, specifically target genes of interest, prior to deep sequencing. MaRS enables researchers to simultaneously collect data on allele frequencies of multiple full-length P. falciparum drug resistance genes (crt, mdr1, k13, dhfr, dhps, and cytochrome b) as well as the mitochondrial genome. Information is captured at the individual patient level for both known and potential new single nucleotide polymorphisms associated with drug resistance. MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistant crt CVIET genotype was observed in 42% of samples, the highly pyrimethamine resistant triple mutant dhps IRN in 92% of samples, and the sulfadoxine resistant dhps SGEAA in 26% of samples. The mdr1 NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistant K13 alleles were identified and 99% of patients carried parasites susceptible to atovaquone-proguanil. Our goal is to implement MaRS at the CDC for routine surveillance of imported malaria cases in the U.S. and aid in the adoption of this system in participating state public health laboratories as well as global partners.

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