Molecular evidence of pfhrp2/pfhrp3 exon 2 deletions and potential HRP2-based RDT diagnostic failure in southern Ethiopia

Deletions of pfhrp2 and pfhrp3 in Plasmodium falciparum compromise the diagnostic performance of HRP2-based rapid diagnostic tests. These deletions can cause false-negative HRP2-based RDT results, leading to missed treatment and ongoing transmission. This study determined the prevalence of pfhrp2/3 gene deletions among febrile patients in Gedeo Zone, southern Ethiopia. A health facility–based cross-sectional study was conducted from October to December 2025 among 384 febrile patients with suspected malaria in Gedeo Zone, southern Ethiopia. Venous blood samples were analyzed using microscopy, SD Bioline RDT, and quantitative polymerase chain reaction (qPCR). Digital polymerase chain reaction (dPCR) was subsequently performed on World Health Organization (WHO)-defined suspected deletion candidates. Among 384 participants, qPCR detected P. falciparum in 29.4% (113/384), P. vivax in 11.7% (45/384), and mixed infections in 2.3% (9/384). Compared with qPCR, SD Bioline RDT demonstrated substantially lower detection rates: 18.0% (69/384) for P. falciparum, 5.5% (21/384) for P. vivax, and 0.5% (2/384) for mixed infections. Among WHO-defined suspected deletion isolates, Pfhrp2 exon 2 deletions were detected in 18.2% (8/44) (95% CI: 8.2–32.7), Pfhrp3 gene deletions in 13.6% (6/44) (95% CI: 5.2–27.4), and dual gene deletions in 11.3% (5/44) (95% CI: 3.8–24.6). Among the WHO-defined suspected deletion candidates, pfhrp2/3 exon 2 deletion was detected in 11.3% (5/44), indicating reduced reliability of PfHRP2-based RDTs in the study area. These findings support the urgent implementation of alternative non-HRP2-based malaria diagnostic strategies in southern Ethiopia.