Limited Polymorphism in Plasmodium falciparum Artemisinin Resistance Kelch13-Propeller Gene Among Clinical Isolates from Bushenyi District, Uganda

Josephat Nyabayo Maniga,1 Saheed Adekunle Akinola,1,2 Martin Odoki,1 John Odda,3,4 Ismail Abiola Adebayo1 1Department of Microbiology and Immunology, Kampala International University Western Campus, Bushenyi, Uganda; 2Faculty of Natural and Agricultural Sciences, North- West University, Mmabatho, South Africa; 3Department of Pharmacology and Toxicology, Kampala International University Western Campus, Bushenyi, Uganda; 4Department of Pharmacology and Therapeutics, Makerere University, Kampala, UgandaCorrespondence: Saheed Adekunle AkinolaDepartment of Microbiology and Immunology, Kampala International University Western Campus, P.O BOX 71, Bushenyi, UgandaTel +256752887872Email saheed.akinola@kiu.ac.ugIntroduction: Drug resistance remains a major challenge in malaria treatment, especially after the emergence of resistance to artemisinin-based combined therapies. Plasmodium falciparum Kelch13 gene mutations are implicated in conferring artemisinin resistance. Thus, this study was aimed at determining the occurrence of Kelch13 (K13) propeller resistance gene polymorphism mutations in Bushenyi district, Uganda.Methods: Participants suspected to have malaria were recruited. P. falciparum was confirmed using antigen histidine-rich protein 2 (HRP2) (Pf) (Access Bio, Inc, USA) and microscopy. Malaria-positive patients were treated with artemeter-lumefantrine (AL). Blood was withdrawn from participants who tested positive for parasites after day 3 and kept in blood filter papers (ET31CHR; Whatman Limited, Kent, UK). DNA was extracted using chelex-suspension method. Nested polymerase chain reaction (PCR) was conducted and the second-round products sequenced using Sanger’s method. Sequenced products were analyzed using DNAsp 5.10.01 software and then blasted on to the NCBI for K13-propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST).Results: Out of 283 enrolled participants, 194 completed the follow-up schedule. A total of 134 (69%) had no parasites on day 3, while 60 (31%) had parasites on that day. Out of the 60 samples, 40 (62%) were positively amplified as P. falciparum, with polymorphisms in the K13-propeller gene detected in 3 (7.5%) out of the 40 amplicons. Polymorphisms at codon 1929, 1788 and 1801 were detected separately in one sample each. Sequences have been deposited in NCBI with accession numbers PRJNA720348 and PRJNA720800.Conclusion: Polymorphisms in the K13-propeller gene previously reported to be associated with artemisinin resistance were not detected in the P. falciparum isolates from Bushenyi district, Uganda. More studies need to be conducted on the new mutations detected so as to understand their association, if any, with ACT resistance.Keywords: artemisinin, resistance, Kelch13, polymorphism