Pooled amplicon sequencing for characterizing mutations in the praziquantel molecular target TRPM _PZQ in schistosome populations from Western Kenya

Mass drug administration (MDA) using Praziquantel is central to efforts to eliminate Schistosomiasis. However, regions which respond poorly to MDA (“persistent hotspots”) have been reported in many regions of Africa, including in Western Kenya. One possible explanation for persistent hotspots is that these areas contain PZQ resistant schistosome parasites. Recent studies have shown that Sm.TRPM _PZQ gene is the molecular target for PZQ in schistosome parasites and that mutations in this gene can result in PZQ resistance. This study characterized mutations within Sm.TRPM _PZQ in 23,420 miracidia collected from both hotspot and non-hotspot villages in Siaya County, western Kenya. We collected triplicate pools of 780.67 (SD ± 183.47) miracidia from 135 people in five hotspot villages, where S. mansoni prevalence remains high despite over 5 annual treatments, and from 62 people from 5 non-hotspots villages where annual treatment resulted in reduction in prevalence. We extracted DNA from each miracidia pool, amplified 15 amplicons covering 1,695bp of the Sm.TRPM _PZQ transmembrane domain and sequenced these to high read depth (21,110x) using a Miseq at KEMRI-CGHR. We identified five high confidence (frequency ≥ 0.01) Sm.TRPM _PZQ variants. These included four synonymous changes and a non-synonymous variant (p.L1476I). p.L1476I is found at similar frequency in non-hotspot (0.040 ± 0.006) and hotspot villages (0.044 ± 0.0050) (Mann Whitney U=18, p= 0.31) and does not impact PZQ-response in Ca ²⁺ reporter assays. Our studies show that resistance variants in Sm.TRPM _PZQ are rare or non-existent in the locations studied and do not explain the existence of hotspots in this region.