Complexity of Infection and <i>Plasmodium falciparum</i> 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 <i>Plasmodium falciparum</i> 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 <i>P. falciparum</i>-positive dried blood spots collected in Mapoussere, Kaele Health District (2022-2023). Using the 4CAST amplicon sequencing assay, we targeted four genes: <i>csp</i> (circumsporozoite protein), <i>ama</i> (apical membrane antigen 1), <i>sera2</i> (serine repeat antigen 2), and <i>trap</i> (thrombospondin-related anonymous protein). Haplotypes were identified using SeekDeep, and diversity metrics including heterozygosity (He), nucleotide diversity ( <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mi>π</mml:mi></mml:math> ), and selection statistics (Tajima's D, Fu and Li's D*, F*) were computed. We successfully genotyped <i>csp</i> in 35% of samples, identifying 22 haplotypes (He = 0.908; <mml:math xmlns:mml="http://www.w3.org/1998/Math/MathML"><mml:mi>π</mml:mi> <mml:mo>=</mml:mo> <mml:mn>0.021</mml:mn></mml:math> ). The vaccine-matched haplotype was present in 20% of genotyped infections. The T cell TH2 and TH3 epitopes of <i>csp</i> showed signs of balancing selection. Both <i>ama</i> and <i>sera2</i> exhibited higher genotyping success and diversity, with <i>ama</i> showing significant Tajima's D values. COI was highest for <i>ama</i> (mean COI = 2.8), followed by <i>sera2</i> (2.1), <i>csp</i> (1.3), and <i>trap</i> (1.2). This study provides a baseline of <i>P. falciparum</i> antigenic diversity and COI in a vaccine-targeted region. The presence of vaccine-matched strains and high diversity in TH2/TH3 epitopes in <i>csp</i> may influence vaccine efficacy. Continued molecular surveillance is essential to monitor antigenic shifts and guide future strategies.