Plasmodium vivax and Plasmodium falciparum mixed infections in human and mosquito hosts: The impact of multi-species infection on parasite densities and transmission to mosquitoes

In co-endemic regions, mixed Plasmodium vivax (Pv) and Plasmodium falciparum (Pf) infections are commonly reported. How mixed species infections compare to single species infections in terms of parasite densities and transmission to mosquitoes is poorly understood. Parasitemia, gametocytemia, and mosquito infectivity were evaluated among Pv mono-infections (n = 284), Pf mono-infections (n = 150), and mixed-Pv-Pf infections (n = 77) recruited at four Ethiopian health facilities. Parasitemia and gametocytemia were quantified in patient blood samples by qPCR. Mosquito infectivity was assessed using direct membrane feeding assays (DMFA), with Plasmodium species confirmation by quantitative PCR. Pf gametocyte prevalence was lower in mixed-infections (64.5%, 49/76) compared with mono-infections (91.8%, 135/147). Gametocyte density among positive carriers was also lower in mixed-infections (p < 0.001). Pv gametocyte prevalence was similar in mono- and mixed-infections despite lower asexual parasite density in mixed infections (p < 0.001). Statistically significant positive correlations between asexual parasitemia and gametocytemia were observed in mono-infections (p < 0.001), but not in mixed-species infections (p = 0.120 for Pv; and p = 0.570 for Pf). The overall proportion of infectious feeds to mosquitoes was high across infection types. Among mixed Pv-Pf parasite carriers, 56.3% (27/48) transmitted both species, often with both species being transmitted to individual mosquitoes. Species-specific gametocyte density remained associated with mosquito infection rates, and these associations were not altered by concurrent infection with the other species. Thus, mixed-species infections were associated with altered asexual and gametocyte densities, notably reduced gametocyte biomass, while mosquito infectivity was not reduced. Because mixed infections are often undetected, they represent a hidden risk for sustaining malaria transmission.