Impact of FY*X homozygosity of the DARC receptor on Plasmodium knowlesi line A1-H.1 invasion.

The Duffy antigen receptor for chemokines (DARC) on the human red blood cell (RBC) surface is a key receptor for Plasmodium vivax and Plasmodium knowlesi invasion. Non-synonymous polymorphisms in the DARC transcriptional unit generate two codominant alleles, FY*A (FY*01) and FY*B (FY*02), which confer Duffy-positive phenotypes, and the FY*ES (FY*02N.01), which in homozygosity results in a Duffy-negative phenotype that impairs RBC invasion by both species. A less frequent variant, the FY*X (FY*02W.01) allele, reduces DARC abundance on the RBC membrane and is predominantly found in Caucasian populations. Its impact on P. knowlesi RBC invasion remains poorly investigated. Here, we measured the impact of FY*X homozygosity on the human-adapted P. knowlesi line A1-H.1 invasion. Duffy phenotypes of seven blood donors were determined using agglutination-based assays, and targeted sequencing confirmed FY*X homozygosity in one donor. Plasmodium knowlesi invasion was quantified in RBCs from donors with different Duffy types in a single experiment. After one invasion cycle, fold multiplication was markedly reduced in FY*X homozygous RBCs (1.5) compared to Duffy-positive donors (5.1-6.5). These findings demonstrate that, despite its low frequency, the FY*X allele substantially limits RBC invasion of P. knowlesi in vitro, underscoring the need for accurate Duffy typing in culture-based studies.