Complexity of Infection and Plasmodium falciparum Circumsporozoite Protein Diversity Prior to Malaria Vaccine Implementation in Kaelé Health District, Cameroon, 2022-2023.

Malaria remains a major public health concern in Cameroon, with Plasmodium falciparum responsible for most morbidity and mortality, particularly among children under five. In response to rising cases, Cameroon began implementing the RTS,S/AS01 malaria vaccine in early 2024. Given the vaccine's strain-specific efficacy, understanding antigenic diversity and complexity of infection (COI) is critical for evaluating long-term impact. We analyzed 100 P. falciparum-positive dried blood spots collected in Mapoussere, Kaele Health District (2022-2023). Using the 4CAST amplicon sequencing assay, we targeted four genes: csp (circumsporozoite protein), ama (apical membrane antigen 1), sera2 (serine repeat antigen 2), and trap (thrombospondin-related anonymous protein). Haplotypes were identified using SeekDeep, and diversity metrics including heterozygosity (He), nucleotide diversity ( π ), and selection statistics (Tajima's D, Fu and Li's D*, F*) were computed. We successfully genotyped csp in 35% of samples, identifying 22 haplotypes (He = 0.908; π = 0.021 ). The vaccine-matched haplotype was present in 20% of genotyped infections. The T cell TH2 and TH3 epitopes of csp showed signs of balancing selection. Both ama and sera2 exhibited higher genotyping success and diversity, with ama showing significant Tajima's D values. COI was highest for ama (mean COI = 2.8), followed by sera2 (2.1), csp (1.3), and trap (1.2). This study provides a baseline of P. falciparum antigenic diversity and COI in a vaccine-targeted region. The presence of vaccine-matched strains and high diversity in TH2/TH3 epitopes in csp may influence vaccine efficacy. Continued molecular surveillance is essential to monitor antigenic shifts and guide future strategies.