Biochemical Evolution of Drug Resistance in Plasmodium falciparum: Molecular Surveillance and Mechanistic Analysis in Three Nigerian Geopolitical Zones

Antimalarial drug resistance in Plasmodium falciparum poses an escalating threat to malaria control in Nigeria, the world's highest burden country, and understanding the molecular evolution and biochemical mechanisms of emerging resistance requires systematic surveillance linked to mechanistic analysis of resistance-associated mutations. This dissertation combines molecular epidemiological surveillance with biochemical mechanistic analysis of drug resistance evolution in P. falciparum across three Nigerian geopolitical zones. Dried blood spot samples from two thousand and four hundred malaria patients at twelve sentinel sites in the South South, North Central, and North West zones were collected over three years. Molecular surveillance targeted resistance-associated mutations in pfcrt, pfmdr1, pfkelch13, pfdhfr, pfdhps, and pfplasmepsin2-3 using pyrosequencing, Sanger sequencing, and targeted deep sequencing. Population genomics approaches were applied to reconstructed haplotypes to estimate resistance allele selection dynamics and evidence for recent selective sweeps. The biochemical mechanisms of clinically emerging resistance phenotypes were characterised using artemisinin-tolerant P. falciparum isolates with partial pfkelch13 mutations cultivated in ex vivo drug sensitivity assays. Haem detoxification by beta-haematin formation rate assays, ring stage survival assays, and oxidative stress response profiling were conducted in sensitive versus tolerant isolates. Surveillance confirms high prevalence of chloroquine resistance-associated pfcrt 76T in all zones and increasing frequency of pfkelch13 mutations associated with artemisinin partial resistance in the North West zone. Mechanistic analysis of emerging kelch13 variants reveals enhanced ring stage antioxidant responses and altered haem detoxification kinetics compared to sensitive parasites. Population genomic analysis identifies three distinct resistance evolution trajectories across zones. Keywords: malaria drug resistance, Plasmodium falciparum, molecular surveillance, biochemical mechanisms, Nigeria.