Recent antimalarial treatment predicts positive HRP2 rapid diagnostic tests in children with no parasites detectable by PCR: direct evidence of antigen persistence from the 2024–25 Uganda Malaria Indicator Survey

Abstract Background Malaria rapid diagnostic tests (RDTs) detecting histidine rich protein 2 (HRP2) underpin case management and national prevalence estimation across sub Saharan Africa. HRP2 persists for weeks after parasite clearance, so a positive RDT may reflect a recently treated infection rather than a current one. Recent nationally representative and multi site studies have reported a high burden of false positive RDT results but were explicitly unable to investigate their causes, lacking either treatment histories or a molecular reference standard. We addressed both gaps. Methods We analysed the 2024–25 Uganda Malaria Indicator Survey, in which children aged 6 to 59 months were tested for malaria by HRP2 based RDT, light microscopy and real time PCR. Weighted prevalence was estimated for each method, accounting for the complex survey design. Diagnostic accuracy of the RDT was assessed against PCR as the primary reference standard and microscopy as a secondary comparator. Records were linked to the survey's children's questionnaire dataset to obtain fever and antimalarial drug use in the two weeks preceding the survey. The primary analysis tested, among children who were PCR negative, whether recent antimalarial use predicted a positive RDT, using survey weighted logistic regression. Results Among 6,975 children, weighted parasite prevalence was 17.5% (95% confidence interval (CI) 15.6 to 19.4) by PCR, 21.1% (95% CI 19.0 to 23.3) by RDT and 12.5% (95% CI 10.9 to 14.1) by microscopy. Against PCR, RDT sensitivity was 79.6% (95% CI 77.3 to 81.8), specificity 89.7% (95% CI 88.9 to 90.5) and positive predictive value 64.1% (95% CI 61.7 to 66.4). Of 794 children positive by RDT but negative by microscopy (792 with a valid PCR result), 232 (29.3%) were PCR positive, indicating genuine submicroscopic infection missed by microscopy rather than RDT error; the remaining 560 (70.7%) were PCR negative. Among PCR negative children, 22.9% of those who had taken an antimalarial in the previous two weeks were RDT positive, compared with 6.5% of those who had not. After adjustment for fever, age, sex, residence, wealth, region and insecticide treated net use, recent antimalarial use remained independently associated with a positive RDT in PCR negative children (adjusted odds ratio (aOR) 1.97, 95% CI 1.33 to 2.93). Recent fever was not independently associated (aOR 1.41, 95% CI 0.95 to 2.10). Conclusions In children with no parasites detectable by PCR, recent antimalarial treatment doubled the odds of a positive HRP2 RDT. Earlier clinical studies inferred antigen persistence from comparisons against microscopy, which cannot exclude submicroscopic infection; by using a molecular reference standard in a national survey we show the association holds, and is larger, where parasitaemia detectable by PCR is excluded. Separately, microscopy missed a substantial number of PCR confirmed infections, cautioning against its use as a sole gold standard. Treatment history should inform interpretation of a positive HRP2 result, and RDT derived prevalence estimates should not be treated as interchangeable with those from other methods.