Non-falciparum malaria and the blind spot of HRP2 based rapid diagnostic tests: a nationally representative PCR study of children in the 2024–25 Uganda Malaria Indicator Survey

Background Malaria diagnosis in Uganda rests almost entirely on rapid diagnostic tests (RDTs) detecting histidine rich protein 2 (HRP2), an antigen expressed only by Plasmodium falciparum . Infections with non-falciparum species are therefore invisible to the national testing algorithm by design. Using a subsample of 466 children from the 2013–14 Democratic Republic of the Congo Demographic and Health Survey, Doctor and colleagues concluded that the risk of missing infections through falciparum-specific RDT use was low, but that conclusion rested on only eight non-falciparum mono-infections. No nationally representative estimate exists for Uganda. Methods We analysed the 2024–25 Uganda Malaria Indicator Survey, in which children aged 6 to 59 months were tested by HRP2 based RDT and by real time PCR with species determination. We estimated survey-weighted prevalence of each species, alone and in mixed infection, and calculated RDT sensitivity separately for P. falciparum mono-infections, mixed infections containing P. falciparum , and non-falciparum mono-infections. Results Among 6975 children, weighted parasite prevalence by PCR was 17.5% (95% confidence interval (CI) 15.6 to 19.4). Non-falciparum species were involved in 2.72% (95% CI 2.25 to 3.19) of children and were the sole species in 1.03% (95% CI 0.75 to 1.30). Weighted prevalence of mono-infection was 0.69% (95% CI 0.47 to 0.90) for P. malariae , 0.30% (95% CI 0.14 to 0.46) for P. ovale , and 4 of 6,975 children (exact 95% CI 0.016 to 0.147%) for P. vivax . RDT sensitivity was 82.3% (95% CI 80.0 to 84.6) for P. falciparum mono-infections and 90.7% (95% CI 84.6 to 95.0) for mixed infections containing P. falciparum , but only 14.3% (95% CI 7.1 to 24.7) for non-falciparum mono-infections. Of 267 PCR confirmed infections missed by the RDT, 60 (22.5%) were non-falciparum mono-infections. Conclusions Approximately one Ugandan child in a hundred carries a malaria infection that HRP2 based RDTs cannot detect, and such infections account for more than a fifth of all infections the RDT misses. Our estimates confirm, with nearly nine times as many events, the direction of previous findings from the Democratic Republic of the Congo, but they place the absolute burden in a national context that the earlier subsample could not support. This is a structural limitation of the diagnostic, not a failure of test quality, and it will not be resolved by improving HRP2 test performance.