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ABSTRACTThe molecular basis of sulfadoxine-pyrimethamine (SP) resistance lies in a combination of single-nucleotide polymorphisms (SNPs) in two genes coding for Plasmodium falciparumdihydrofolate reductase (Pfdhfr) and P. falciparumdihydropteroate synthase (Pfdhps), targeted by pyrimethamine and sulfadoxine, respectively. The continued use of SP for intermittent preventive treatment in pregnant women in many African countries, despite SP's discontinuation as a first-line antimalarial treatment option due to high levels of drug resistance, may further increase the prevalence of SP-resistant parasites and/or lead to the selection of new mutations. An antimalarial drug resistance surveillance study was conducted in western Kenya between 2010 and 2013. A total of 203 clinical samples from children with uncomplicated malaria were genotyped for SNPs associated with SP resistance. The prevalence of the triple-mutant PfdhfrC50I51R59N108I164genotype and the double-mutant PfdhpsS436G437E540A581A613genotype was high. Two triple-mutant Pfdhpsgenotypes, S436G437E540G581A613and H436G437E540A581A613, were found, with the latter thus far being uniquely found in western Kenya. The prevalence of the S436G437E540G581A613genotype was low. However, a steady increase in the prevalence of the Pfdhpstriple-mutant H436G437E540A581A613genotype has been observed since its appearance in early 2000. Isolates with these genotypes shared substantial microsatellite haplotypes with the most common double-mutant allele, suggesting that this triple-mutant allele may have evolved locally. Overall, these findings show that the prevalence of the H436G437E540A581A613triple mutant may be increasing in this population and could compromise the efficacy of SP for intermittent preventive treatment in pregnant women if it increases the resistance threshold further.

ABSTRACTThe 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. falciparumdrug resistance genes (crt, mdr1, k13, dhfr, dhps, and the cytochrome bgene), 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. The MaRS pipeline was validated using 245 imported malaria cases that were reported to the Centers for Disease Control and Prevention (CDC). The chloroquine resistance crtCVIETgenotype (mutations underlined) was observed in 42% of samples, the highly pyrimethamine-resistant dhpsIRNtriple mutant in 92% of samples, and the sulfadoxine resistance dhpsmutation SGEAA in 26% of samples. The mdr1NFSND genotype was found in 40% of samples. With the exception of two cases imported from Cambodia, no artemisinin resistance k13alleles 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 United States and to aid in the adoption of this system at participating state public health laboratories, as well as by global partners.