High prevalence of mixed Plasmodium falciparum and Plasmodium vivax co-infections in Duffy negative individuals in Ethiopia.

Malaria remains a major public health concern in Ethiopia, where both Plasmodium falciparum and Plasmodium vivax are endemic. Notably, P.vivax infections have been documented in duffy-negative individuals, who were traditionally thought to be resistant to this parasite. PvDBP gene duplication has been proposed as a mechanism for P. vivax to invade erythrocytes in these individuals, but evidence is limited. We conducted a cross-sectional study from 2020 to 2021 with1,154 febrile patients in three Ethiopian regions: Gambela, Oromia, and SNNPR. Plasmodium species were identified by PCR, Duffy genotypes by TaqMan allelic discrimination assay and PvDBP gene duplication by semi-nested PCR. Plasmodium infection was detected in 46% of patients: 49% had P. falciparum, 26% had mixed P.falciparum/ P.vivax infections, and 23% had P. vivax mono-infections. Duffy genotyping showed 41% were Duffy negative, 44% heterozygous, and 16% Duffy positive homozygous, with significant regional differences. As expected, P. vivax mono-infections were rare among Duffy-negative individuals (2.4%), while P. falciparum infection prevalence was similar across Duffy genotypes. Among P. falciparum/P. vivax mixed-infections, 25% were Duffy negative. Off 155 samples successfully tested for the PvDBP gene duplication, 78% showed gene duplication, especially in Gambela (94%), where Duffy-negative individuals were most prominent. The prevalence of duplication did not differ between Duffy-positive homozygous and heterozygous individuals. Only one P. vivax mono-infection in a Duffy negative individual was examined and no gene duplication was detected. Our findings suggest possible interactions between Duffy negative infection by P. vivax and co-infection with P. falciparum. The high prevalence of PvDBP gene duplication, particularly in regions with high Duffy negativity, may reflect evolutionary pressure to overcome host receptor barriers. These results underscore the need for molecular surveillance to monitor emerging parasite adaptations in populations previously considered resistant to P. vivax.