Novel Plasmodium falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of artemisinin-based combination therapy deployment

Josephat Nyabayo Maniga, Mong’are Samuel, Odda John, Masai Rael, Jacqueline Njeri Muchiri, Pacifica Bwogo, Odoki Martin, Vidya Sankarapandian, Mfitundinda Wilberforce, Ochweri Albert, Sarah Kemuma Onkoba, Ismail Abiola Adebayo, Rasheed Omotayo Adeyemo, Saheed Adekunle Akinola

Currently, chemotherapy stands out as the major malaria intervention strategy, however, anti-malarial resistance may hamper global elimination programs. Artemisinin-based combination therapy (ACT) stands as the drug of choice for the treatment of Plasmodium falciparum malaria. Plasmodium falciparum kelch13 gene mutations are associated with artemisinin resistance. Thus, this study was aimed at evaluating the circulation of P. falciparum k13 gene polymorphisms from Kisii County, Kenya during an era of ACT deployment. Participants suspected to have malaria were recruited. Plasmodium falciparum was confirmed using the microscopy method. Malaria-positive patients were treated with artemether-lumefantrine (AL). Blood from participants who tested positive for parasites after day 3 was kept on filter papers. DNA was extracted using chelex-suspension method. A nested polymerase chain reaction (PCR) was conducted and the second-round products were sequenced using the Sanger method. Sequenced products were analysed using DNAsp 5.10.01 software and then blasted on the NCBI for k13 propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST). To assess the selection pressure in P. falciparum parasite population, Tajima' D statistic and Fu & Li's D test in DnaSP software 5.10.01 was used. Out of 275 enrolled participants, 231 completed the follow-up schedule. 13 (5.6%) had parasites on day 28 hence characterized for recrudescence. Out of the 13 samples suspected of recrudescence, 5 (38%) samples were positively amplified as P. falciparum, with polymorphisms in the k13-propeller gene detected. Polymorphisms detected in this study includes R539T, N458T, R561H, N431S and A671V, respectively. The sequences have been deposited in NCBI with bio-project number PRJNA885380 and accession numbers SAMN31087434, SAMN31087433, SAMN31087432, SAMN31087431 and SAMN31087430 respectively. WHO validated polymorphisms in the k13-propeller gene previously reported to be associated with ACT resistance were not detected in the P. falciparum isolates from Kisii County, Kenya. However, some previously reported un-validated k13 resistant single nucleotide polymorphisms were reported in this study but with limited occurrences. The study has also reported new SNPs. More studies need to be carried out in the entire country to understand the association of reported mutations if any, with ACT resistance.