screened for resistance SNPs at the following gene targets by pyrosequencing: PfATPase6 (atpase6), chloroquine resistance transporter (pfcrt), cytochrome b (cytb), dihydrofolate reductase-thymidylate synthase (dhfr), dihydropteroate synthetase (dhps), and multidrug resistance protein (mdr1). Isolates were also screened for mutations of the kelch13 propeller region (kelch13) by Sanger sequencing. Mutation prevalence was calculated and compared across time periods.Results: Two-hundred, twenty-three unique isolates of P. falciparum were identified from 2008-2009 (n = 75), 2013-2014 (n = 79) and 2017-2018 (n = 69). Of 223 isolates, 126 had a documented travel history, with most (88%) imported from sub-Saharan Africa. Significant decreases in mutant genotypes from 2008-09 to 2013-14 for mdr1 N86Y (p < 0.001), D1246Y (p = 0.007), pfcrt K76T (p = 0.032) were observed. Similarly, significant decreases in mutant allelic frequencies were observed from 2013-14 to 2017-18 for mdr1 N1042D (p = 0.0065); and cytB Y268NSC (p = 0.0038) and mdr1 N86Y (p = 0.003) across the three time frames. Significant increases in mutant allelic frequencies for pfcrt C72S (p = 0.027); dhfr A16 V (p = 0.01), C50R (p = 0.04); dhps K540E (p < 0.001), A581G (p < 0.001), A613S (p < 0.001), and mdr1 N1042D (p < 0.001) were observed between 2008-09 and 2013-14. A significant increase in mutant allelic frequencies for atpase6 was observed from 2013-14 and 2017-18 (p < 0.0001). Kelch13 mutations were not identified in any isolate between 2008-09 and 2013-14, including the 4 imported from southeast Asia.
Conclusion:Mutant genotypes for several molecular markers of drug resistance were highly prevalent among P. falciparum isolates imported to Ontario, especially mutations in dhfr conferring resistance to proguanil. Our observation of minor genotypes confirms the heterogeneous nature of infection, which may lead to differential drug resistance levels and therapeutic failure. Atpase6 has shown significantly increases in mutant allelic frequencies associated with artemisinin resistance and warrants sustained surveillance.
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