Comparative evaluation of a multi-antigen malaria rapid diagnostic test against qPCR and microscopy in a co-endemic area of Southern Ethiopia

Because P. falciparum and P. vivax coexist in the Gedeo Zone, accurate malaria diagnosis is essential for effective case management. Low parasitemia and pfhrp2/3 gene deletions limit the performance of conventional HRP2-based RDTs. The new Bioline Malaria multi-Ag Pf/Pf/Pv RDT, detecting HRP2 and dual pLDH for both species, addresses this limitation. This study evaluates its field performance in detecting low-density infections during peak transmission season using expert microscopy and qPCR as references. A health facility-based cross-sectional study enrolled 579 febrile patients from health facilities in Gedeo Zone during the major transmission period from October to December 2025. Venous blood was tested with the conventional and multi-antigen Bioline RDTs per manufacturer directions, alongside expert microscopy and qPCR. Diagnostic performance parameters were calculated, comparing RDTs to expert microscopy and qPCR. Among 579 febrile patients, malaria prevalence was 36.4% (211/579) by light microscopy, 33.2% (192/579) by multi-antigen RDT, and highest with qPCR 40.8% (236/579), which also detected a greater number of P. falciparum (20.7%), P. vivax (16.9%), and mixed infections (3.3%). Using microscopy as a reference, multi antigen RDT showed high sensitivity 86.2% (95% CI: 80.8-90.6%) and specificity 98.6% (95% CI: 96.8-99.5%) with almost perfect agreement (κ = 0.87); however, against qPCR, sensitivity decreased for P. falciparum 77.1% (95% CI: 71.2-82.3%) and despite consistently high specificity 97.1% (95% CI: 94.7-98.5%), indicating missed submicroscopic infections. The Bioline Malaria Ag Pf/Pf/Pv RDT demonstrated high specificity and substantial agreement with microscopy but moderate sensitivity compared with qPCR, suggesting that low-density infections were overlooked. These findings highlight the need for more sensitive diagnostic techniques for malaria surveillance and control in endemic environments while supporting the clinical utility of RDTs for regular clinical diagnosis.