Molecular 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

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 sub-microscopic infections. 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 (DBS) 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. Logistic regression was used to determine factors associated with sub-microscopic malaria infections. 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. Based on microscopy and real-time PCR, the highest proportion of parasites was detected in children 48–50 months old (15.8% and 21.0%); Children living in rural areas (15.7% and 22.3%); Lango sub-region (31.6% and 43.7%); and Children whose mothers had no education (16.9% and 21.6%) respectively. Real-time PCR confirmed the presence of P. falciparum (85%), P. malariae (4%), P. ovale (2%) and P. vivax (<1%) mono-infections while (9%) were mixed infections. Highest rate of mixed infections was recorded in Lango (18%), Karamoja (14.7%), Acholi (12.8%) and Tooro (9.6%) sub-regions. Sub-microscopic infection was detected in 7% (385) out of the 5,512 that were blood smear negative. Sub-microscopic infection was more common in Lango [aOR 2.52, 95% CI: 1.31–4.87], p = 0.006, less common in Kigezi [aOR 0.14, 95% CI: 0.06–0.36], p < 0.001 and associated with age with the highest recorded in older children 48–59 months old [aOR 2.28, 95% CI: 1.20–4.35], p = 0.012. Urban residence was associated with lower odds of sub-microscopic infection [aOR 0.39, 95% CI: 0.26–0.58], p < 0.001. Overall, molecular-based tools (PCR) detected a higher prevalence of malaria parasitemia than blood smear microscopy, revealed the presence of sub-microscopic infections in microscopy negative individuals and presence of non-P. falciparum species across all survey regions in Uganda. Proportions of sub-microscopic infections varied across the survey regions. These findings suggest the potential for consideration and integration of molecular-based methods in malaria surveys to detect and estimate the burden of parasite reservoir in sub-microscopic infections that is often missed by blood smear microscopy. The observed occurrence and wide geographical spread of non-P. falciparum species may have implication for deployment of diagnostic tools that detect all malaria parasite species occurring in Uganda.