Genomic Surveillance to Estimate the burden of Sub-microscopic malaria infections, Parasite Species Composition and Implication for Elimination in Uganda; First Evidence at National Scale - 2025

Background: Malaria morbidity and mortality declined in Uganda between 2010 and 2020; however, recent reports indicate potential reversal and loss of control gains. Demonstration of the presence of malaria parasites in human populations is an essential parameter for monitoring transmission and progress of interventions in endemic settings; however, surveillance systems heavily rely on blood smear microscopy that often misses submicroscopic infections. Methods: From September 2024 to April 2025, blood samples were collected from children under 5 years old in cross-sectional surveys in all regions of Uganda. Thick blood smears and dried blood spots were collected and analysed with microscopy and real-time PCR respectively. Data were analysed, with STATA and geographical information systems (ArcGIS) and reported as proportions and geospatial maps. Results: Overall, 6298 samples were analysed out of which 819 (13%) and 1,071 (17%) were positive for malaria parasites by microscopy and real-time PCR respectively. Highest proportion of parasites was detected in children 48-50 months old (15.8% and 21.0%) by microscopy and real-time PCR respectively; Children living in rural areas (15.7% and 22.3%); Lango sub-region (31.6% and 43.7%); Children whose mothers had no education (16.9% and 21.6%) by microscopy and real-time PCR respectively. Real-time PCR confirmed the presence of P. falciparum (85%), P. malariae (4%), P. ovale (2%), P. vivax (<1%) and mixed infections (9%). The Lango sub-region recorded the highest rate of mixed infections (18%) followed by Karamoja (14.7%), Acholi (12.8%) and Tooro (9.6%). Among children who tested negative for malaria by microscopy, 7% were positive by real-time PCR. Sub-microscopic infections were most common in Lango (18%), Acholi (13%) and Teso (10.6%) and lowest in Kigezi (1.5%), Ankole (1.7%) and Kampala City (<1%). Conclusion: These findings emphasize potential for consideration and integration of molecular-based methods in malaria surveillance systems to detect sub-microscopic parasite reservoir that is often missed by blood smear microscopy. The observed occurrence and wide geographical spread of non-P. falciparum species across the country suggests the need for deployment of diagnostic tools that detect all malaria parasite species for routine diagnosis and surveillance in Uganda.