Detection and population genetics of the Plasmodium falciparum dhps 581G variant associated with high-level sulfadoxine resistance in the Democratic Republic of the Congo

Each year, over 125 million women in the Democratic Republic of the Congo (DRC) are at risk for malaria in pregnancy, representing a significant source of morbidity and mortality to both mothers and their children. To prevent malaria in pregnancy, sulfadoxine-pyrimethamine is used throughout sub-Saharan Africa as a safe and effective form of chemoprophylaxis. However, high-level sulfadoxine resistance threatens to abrogate these protective effects, especially the spread of the single nucleotide variant dihydropteroate synthetase (dhps) 581G. To address this concern, we aimed to develop a novel molecular diagnostic capable of detecting this variant and to study the population genetics surrounding it within the DRC. First, we developed a suite of CRISPR-based diagnostics based on the platform SHERLOCK, or Specific High-Sensitivity Enzymatic Reporter UnLOCKing. We developed SHERLOCK assays capable of detecting all Plasmodium species known to cause human malaria and species-specific detection of P. vivax and P. falciparum, the species responsible for the majority of malaria cases worldwide. We found that our suite of Plasmodium SHERLOCK assays achieved low limits of detection in vitro comparable with other molecular diagnostics, and high sensitivity and specificity against a diverse panel of clinical isolates. Next, we developed a SHERLOCK assay capable of detecting the dhps 581G variant. We found that our dhps single nucleotide variant SHERLOCK assay achieved good sensitivity and high specificity when applied to a panel of deep-sequenced clinical samples, with false-negative calls only at low parasite densities. Finally, we conducted whole genome sequencing on a panel of clinical isolates from the Democratic Republic of the Congo, Ghana, and Tanzania to study the impact of the dhps 581G variant on P. falciparum population structure within the DRC. In contrast with other recent work, we did not find significant contribution to population structure from the dhps locus. We then examined the extended haplotype surrounding the dhps locus and found no evidence of a recent hard selective sweep. Rather, we found the dhps 581G variant on multiple extended haplotypes from different locations, consistent with a soft selective sweep at this locus.Doctor of Philosoph