High prevalence of mixed <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> co-infections in Duffy negative individuals in Ethiopia

Malaria remains a major public health concern in Ethiopia, where both <i>Plasmodium falciparum</i> and <i>Plasmodium vivax</i> are endemic. Notably, <i>P.vivax</i> infections have been documented in duffy-negative individuals, who were traditionally thought to be resistant to this parasite. <i>PvDBP</i> gene duplication has been proposed as a mechanism for P<i>. vivax</i> 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. <i>Plasmodium</i> species were identified by PCR, Duffy genotypes by TaqMan allelic discrimination assay and <i>PvDBP</i> gene duplication by semi-nested PCR. <i>Plasmodium</i> infection was detected in 46% of patients: 49% had <i>P. falciparum</i>, 26% had mixed <i>P.falciparum</i>/ <i>P.vivax</i> infections, and 23% had <i>P. vivax</i> mono-infections. Duffy genotyping showed 41% were Duffy negative, 44% heterozygous, and 16% Duffy positive homozygous, with significant regional differences. As expected, <i>P. vivax</i> mono-infections were rare among Duffy-negative individuals (2.4%), while <i>P. falciparum</i> infection prevalence was similar across Duffy genotypes. Among <i>P. falciparum</i>/<i>P. vivax</i> mixed-infections, 25% were Duffy negative. Off 155 samples successfully tested for the <i>PvDBP</i> 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 <i>P. vivax</i> 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 <i>P. vivax</i> and co-infection with <i>P. falciparum</i>. The high prevalence of <i>PvDBP</i> 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 <i>P. vivax</i>.