Point of care performance of the improved BIOCREDIT Ag Pf/Pv (pLDH/pLDH) rapid diagnostic test: a cross-sectional study at a health center setting in Ethiopia

BACKGROUND: In Ethiopia, co-endemic Plasmodium falciparum and P. vivax require species-specific point-of-care diagnosis and treatment. Following pfhrp2/3 deletions, Ethiopia adopted non-HRP2/3 rapid diagnostic tests (RDTs). This study evaluated the point-of-care diagnostic performance of the improved BIOCREDIT Ag Pf/Pv (pLDH/pLDH) RDT compared to microscopy (comparator), using the 18S rRNA-based qPCR as the reference standard. METHODS: This prospective cross-sectional study was nested within an operational feasibility study of radical cure for P. vivax with tafenoquine or primaquine after semi-quantitative G6PD testing in Ethiopia. Conducted from December 2024 to September 2025, it took place at Village 8/9 and Beto Health Centers in Gambella and South Ethiopia regions, respectively. Self-reporting clinical malaria cases (n = 1200) were enrolled, with 846 from Village 8/9 and 354 from Beto. Approximately 150 µL of capillary blood was collected from each participant for point-of-care RDTs (5 µL), thick and thin smear microscopy (7 µL), and dried blood spots (140 µL) for 18S rRNA qPCR. Diagnostic performance was evaluated through sensitivity, specificity, kappa agreement (κ), and case detection comparisons across RDT, qPCR, and microscopy. Associations between parasite density and detectability, as well as the geographic distribution of species, age, and sex, were assessed. RESULTS: Using 18S qPCR, malaria prevalence rate was 40.6% (487/1,200): 63.0% P. falciparum, 29.6% P. vivax, and 7.4% mixed infections. P. falciparum predominated in Gambella (68.5%, 217/317) vis-à-vis South Ethiopia (52.9%, 90/170), whereas P. vivax was more common in South Ethiopia (37.6%, 64/170) vis-à-vis Gambella (25.2%, 80/317). The RDT showed higher sensitivity for qPCR-confirmed Plasmodium infections (89.7%) than routine microscopy (59.8%), with comparable specificities (97.2% versus 96.5%). Species-specific sensitivities were 87.9% for P. falciparum, 86.7% for P. vivax, and 50.0% for mixed infections. Agreement with qPCR was strong for RDT (κ = 0.92) and moderate for microscopy (κ = 0.79). Overall diagnostic accuracy was excellent (AUC ≥ 0.92 for both species). RDT sensitivity declined at low parasite densities but reached 100% at ≥ 5,000 copies/µL for both species. CONCLUSIONS: The RDT demonstrated strong accuracy for point-of-care, species-specific malaria diagnosis in Ethiopian primary healthcare. Its high sensitivity, particularly for moderate- to high-density infections, supports routine clinical use following Ethiopia's shift to non-HRP2 RDTs.