bioRxiv2026
Background Malaria remains a major public health challenge in Cameroon, with sustained transmission particularly in forest-savannah transition zones. Although Plasmodium falciparum is the predominant malaria parasite, non-falciparum species such as P. malariae also contribute to the human infectious reservoir and to onward transmission. Parasite genetic diversity and multiplicity of infection (MOI) are recognized determinants of malaria transmission and may influence the prevalence and intensity of infection in mosquito vectors. However, whether parasite genetic diversity and MOI differentially influence the infectivity of P. falciparum and P. malariae to local Anopheles coluzzii populations remains poorly characterized. This knowledge gap is particularly relevant in the Mfou Health District, where malaria transmission persists and both P. falciparum and non-falciparum infections occur. Methods A cross-sectional survey was conducted between September 2022 and November 2023 in twelve primary schools in the Mfou Health District. Finger-prick blood samples were collected from children aged 3-17 years and examined by microscopy to identify Plasmodium infections and gametocyte carriers. Gametocyte-positive participants were enrolled in direct membrane feeding assays (DMFA) using locally reared Anopheles coluzzii. Blood samples were tested under two conditions: whole blood (WB) and replacement with naive AB serum. Mosquito infection outcomes were assessed at the oocyst (day 7) and sporozoite stages (day 14). Parasite species were confirmed by quantitative PCR (qPCR), and genetic diversity was assessed using seven microsatellite loci, together with msp1 and msp2 markers for P. falciparum and pmmsp1 for P. malariae. MOI and expected heterozygosity (He) were calculated, and their associations with mosquito infection prevalence and intensity were evaluated using generalized linear models. Results Among 2,807 children screened, 2,111 (75.2%) were infected with Plasmodium, and 388 (18.4%) carried gametocytes. A total of 169 children aged 4-13 years were selected for membrane feeding assays using Anopheles coluzzii, of whom 68% successfully infected mosquitoes. Among 10,872 dissected mosquitoes, 20.2% were infected at the oocyst stage, with both infection prevalence and intensity being higher in mosquitoes fed on AB serum-reconstituted blood than with whole blood. Sporozoite-stage infections were detected in 5.8% of mosquitoes, predominantly under AB serum conditions. Mosquito infection prevalence and intensity increased with gametocyte density, although submicroscopic gametocyte carriers were also infectious. P. falciparum exhibited high genetic diversity (MOI 2.4 {+/-} 0.8; He 0.72 {+/-} 0.07), while P. malariae showed limited diversity (MOI 0.11-0.21; He {approx} 0). Mixed-species and polyclonal infections were associated with enhanced infection outcomes. Moreover, the msp2 FC27 allelic family in P. falciparum and the 800-bp pmmsp1 allele in P. malariae were associated with oocyst development. Overall, parasite genetic diversity, multiplicity of infection, and species composition were positively associated with mosquito infectivity, with only minor seasonal variation observed. Conclusion Malaria transmission in Mfou is sustained by genetically diverse and multiclonal P. falciparum infections, with P. malariae also contributing to the human infectious reservoir. The association of parasite genetic diversity, MOI, and allele-specific factors with mosquito infectivity underscores the importance of addressing asymptomatic and multiclonal infections in malaria control strategies.