National PCR survey finds sub-microscopic malaria in 7% of microscopy-negative Ugandan children under five

A national cross-sectional study of children under five in Uganda found that real-time PCR detected malaria parasites in 17% of 6,298 samples analysed, compared with 13% detected by blood-smear microscopy. The survey, conducted from September 2024 to April 2025 across all regions, also identified Plasmodium species beyond P. falciparum and documented substantial geographic variation in sub-microscopic infections. The findings indicate that microscopy alone may not capture the full extent of malaria parasitaemia in the surveyed population.

Investigators collected thick blood smears and dried blood spots during cross-sectional surveys. Microscopy assessed the smears, while real-time PCR analysed the dried blood spots. Statistical analysis and geographical information systems were used to calculate proportions and produce geospatial maps, and logistic regression was used to examine factors associated with sub-microscopic malaria infection. Microscopy detected parasites in 819 samples, or 13%, while PCR detected them in 1,071 samples, or 17%. Among the 5,512 children with negative blood smears, 385 had sub-microscopic infections, corresponding to 7% of that microscopy-negative group rather than the full sample.

The highest measured parasite positivity was reported in several groups. Children aged 48–50 months had positivity of 15.8% by microscopy and 21.0% by PCR. Rural children had rates of 15.7% and 22.3%, respectively, while children whose mothers had no education had rates of 16.9% and 21.6%. The Lango sub-region recorded the highest regional proportions, at 31.6% by microscopy and 43.7% by PCR. These descriptive differences did not by themselves establish that residence, age or maternal education caused the observed variation.

PCR identified P. falciparum in 85% of infections, P. malariae in 4%, P. ovale in 2% and P. vivax in less than 1%; mixed infections accounted for 9%. Mixed-infection proportions were highest in Lango at 18%, followed by Karamoja at 14.7%, Acholi at 12.8% and Tooro at 9.6%. In regression analyses, sub-microscopic infection was more common in Lango than the study’s reference area, with an adjusted odds ratio of 2.52 (95% confidence interval 1.31–4.87; p=0.006), and less common in Kigezi, with an adjusted odds ratio of 0.14 (95% CI 0.06–0.36; p<0.001). Children aged 48–59 months also had higher odds than the reference age group, with an adjusted odds ratio of 2.28 (95% CI 1.20–4.35; p=0.012). Urban residence was associated with lower odds, with an adjusted odds ratio of 0.39 (95% CI 0.26–0.58; p<0.001); these were associations rather than causal effects.

The researchers concluded that the findings support considering molecular methods in malaria surveys to identify infections missed by microscopy and estimate the distribution of the parasite reservoir. The presence and broad geographic spread of non-P. falciparum species also indicate implications for diagnostic tools intended to detect all malaria parasite species occurring in Uganda. Interpretation is bounded by the study population and design: the findings concern children under five sampled during one national survey period, and the cross-sectional measurements describe prevalence at that time rather than changes over time or effects of a surveillance intervention.

Subject of Research: National molecular surveillance of sub-microscopic malaria in Ugandan children under five

Article Title: 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

Article References: Agaba, B. B., Katairo, T., Tukwasibwe, S., Makoha, C., Abaasa, C. N., Wiringilimaana, I., Semakuba, F., Nsengimaana, B., Nakasaanya, J., Ssemaganda, A., Ayitewala, A., Nabirwa, G., Adranya, B., Ojaku, A., Nabadda, S., Nabakooza, J., Rukundo, G., Amutuhaire, M., Kimuli, R., … Agaba, E. (2026). 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. BMC Infectious Diseases. https://doi.org/10.1186/s12879-026-14473-2

Image Credits: AI Generated

DOI: 10.1186/s12879-026-14473-2

Keywords: malaria, PCR surveillance, sub-microscopic infection, Uganda, children under five, Plasmodium falciparum, blood-smear microscopy, parasite species, cross-sectional survey, Molecular, surveillance, estimate