Detection of Low-Frequency Artemisinin Resistance Mutations C469Y, P553L and A675V in Asymptomatic Primary School Children in Kenya

Background: To understand the emergence and spread of drug-resistant parasites in malaria-endemic areas, accurate assessment and monitoring of antimalarial drug resistance markers is critical. Recent advances in next-generation sequencing (NGS) technologies have enabled the tracking of drug-resistant malaria parasites. Methods: In this study, we used Targeted Amplicon Deep Sequencing (TADs) to characterise the genetic diversity of the Pfk13 , Pfdhfr , Pfdhps , and Pfmdr1 genes among primary school-going children in 15 counties in Kenya (Bungoma, Busia, Homa Bay, Migori, Kakamega, Kilifi, Kirinyaga, Kisii, Kisumu, Kwale, Siaya, Tana River, Turkana, Vihiga, and West Pokot,). A total of 920 dried blood spot (DBS) samples collected from 121 selected primary schools within the country were used to extract genomic DNA. A nested polymerase chain reaction (PCR) was used to generate amplicons that were sequenced to determine the prevalence of known and novel polymorphisms. Results: The analysis of Pfk13 mutations associated with artemisinin resistance showed that the C469Y mutation was found in 23 samples (4%), the A675V mutation was identified in 2 samples (1.7%), and the P553L mutation was present as a mixed genotype in 7 samples (1.2%), all as mixed infections. The A578S mutation, one of the most common nonsynonymous mutations found in Africa, was also identified in mixed infections, appearing in 15.2% of the 87 samples analysed. The Pfdhfr 51I and 108N pyrimethamine-resistance mutations were at fixation (100% frequency), and the Pfmdr1 Y184F mutation that has been associated with reduced susceptibility to lumefantrine was found in 97.5% of the samples as mixed genotype infections. Conclusion: The genomics surveillance of asymptomatic school children in Kenya provides an early warning signal of at least 1 of the 3 validated artemisinin resistance mutations circulating in all regions in Western Kenya sampled except Homa Bay and Kisii Counties. These signals in asymptomatic, mixed infections would have been missed without deep sequencing.