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 incompletely 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 PCR. Pf gametocyte prevalence was lower in mixed-infections (64.5%, 49/76) compared with mono-infections (91.8%, 135/147); among gametocyte-positive parasite carriers gametocyte density 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). Transmission to mosquitoes was high across infections and infection combinations. Among infectious Pv-Pf parasite carriers, 56.3% (27/48) transmitted both species, often with both species being transmitted to individual mosquitoes. The associations between (species specific) gametocyte density and mosquito infection rates was unaffected by the concurrent presence of the other Plasmodium species. Although mixed-species infections may have different parasite and gametocyte densities compared to mono-infections, we observed no evidence for competition between species in mosquitoes. Because mixed infections are often undetected, they represent a hidden risk for sustaining malaria transmission. Author SummaryMalaria remains a major public health challenge in Ethiopia, where Plasmodium vivax and Plasmodium falciparum often occur together in the same patient. While mixed-species infections are common, little is known about how they compare to single-species infections in terms of parasite levels and transmission to mosquitoes. In this study, we examined patients with P. vivax, P. falciparum, or mixed infections across four health facilities. We measured parasite and gametocyte densities in blood samples and tested whether patients could infect mosquitoes. We found that P. falciparum gametocyte levels were lower in mixed infections compared to single-species infections, while P. vivax gametocyte levels were similar regardless of infection type. Importantly, patients with mixed infections were still highly infectious to mosquitoes, and many transmitted both parasite species simultaneously. These findings show that mixed infections, which are often missed by routine diagnosis, can contribute substantially to malaria transmission. Recognizing and addressing mixed-species infections is therefore critical for malaria control and elimination efforts.