Are malaria rapid diagnostic test results stable over time to support verification of surveillance data?

(2025) 24:356 Ngufor et al. Malaria Journal https://doi.org/10.1186/s12936-025-05595-0 Malaria Journal Open Access RESEARCH Are malaria rapid diagnostic test results stable over time to support verification of surveillance data? Corine Ngufor1*†, Kim A. Lindblade2†, Sunday Atobatele3, Arthur Mpimbaza4, Idelphonse Ahogni1, Nelson Ssewante4, Ese Akpiroroh3, Augustin Kpemasse5, Onyebuchi Okoro6, Bosco Agaba7, Shawna Cooper8, Kevin Griffith9 and Michael Humes9 Abstract Background Rapid diagnostic tests (RDTs) have improved malaria case management by enabling point-of-care confirmation of infection, particularly in low-resource settings. In addition to clinical use, RDT results recorded in health facility registers are a critical component of national malaria surveillance systems. Recently, national programmes have explored using stored RDT cassettes to validate register data. However, manufacturers caution that results should be read within 15–30 min, raising concerns about result validity after this period. This study evaluated the stability of RDT results over a one-month period to assess whether stored cassettes can reliably reflect initial test outcomes. Methods A prospective, observational study was conducted in 48 health facilities across Benin, Nigeria, and Uganda from June to September 2023. A digital artificial intelligence (AI)-powered RDT reader (HealthPulse, Audere, Seattle WA USA) was used to photograph RDTs immediately after interpretation by health workers and again at one week and one month. RDTs were stored under typical health facility conditions during the study. Images were independently interpreted by a trained panel, with results categorized as positive, negative, invalid, or uninterpretable. Only RDTs with valid interpretations at all three time points were included in the final analysis. Positive and negative predictive values (PPV and NPV) were calculated to measure the accuracy of results from stored RDTs relative to the initial interpretation. Results Out of 54,251 RDTs captured, 45,155 (83.2%) met inclusion criteria. At one month, 95.1% of initially positive RDTs remained positive, and 95.3% of initially negative RDTs remained negative. The PPV of a positive result at one month was 96.3% (95% CI 96.1, 96.5), while the NPV of a negative result was 93.8% (95% CI 93.4, 94.1). Most result changes occurred within the first week. Faint lines were associated with higher rates of change in both directions; 26.8% changing from positive to negative and 48.1% changing from negative to positive. Stability of results also varied across RDT products and specific test lines. Conclusions Stored RDT cassettes maintain high result stability over one month and can serve as a reliable reference to verify health facility records. Result changes were linked to premature interpretation, faint lines or product- or linespecific characteristics. Adherence to manufacturer-recommended read times may reduce the proportion of RDTs † Corine Ngufor and Kim A. Lindblade contributed equally to the publication. *Correspondence: Corine Ngufor Full list of author information is available at the end of the article © The Author(s) 2025. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Ngufor et al. Malaria Journal (2025) 24:356 Page 2 of 11 that change from negative to positive. These findings support the utility of stored RDTs in improving data quality and rational antimalarial use in malaria-affected settings. Keywords Malaria, Rapid diagnostic tests, Surveillance, Benin, Nigeria, Uganda, Stability Background Rapid diagnostic tests (RDTs) have transformed malaria case management in resource-limited settings by enabling point-of-care confirmation of infection prior to treatment. They require minimal expertise, no refrigeration or specialized equipment and no electricity, making them suitable for use in a wide range of environments. RDT results recorded in health facility registers also underpin malaria surveillance systems in endemic countries, providing essential data for tracking disease trends, guiding resource allocation, and assessing the effectiveness of control measures [1]. Malaria RDTs are lateral flow immunoassays that use antibodies to detect specific antigens produced by malaria parasites in the bloodstream. These antibodies are immobilized on a nitrocellulose strip housed within a plastic cassette. A few drops of blood from a finger prick are placed into one well of the cassette and buffer solution is added to a second well. The buffer lyses red blood cells, releasing any parasite proteins. Dye-labelled antibodies specific for one or more Plasmodium species then bind to parasite antigens. Capillary action moves the blood and antigen–antibody complexes along the membrane, where they are captured by one or more lines of fixed antibodies (the T, or test, lines), forming visible colored bands in the results window. The control (C) line, located further down the membrane, captures excess dye-labelled antibodies and forms a visible colored band that indicates the test has functioned correctly. Malaria RDTs primarily target two antigens: histidinerich protein 2 (HRP2) and Plasmodium lactate dehydrogenase (pLDH), with aldolase used less commonly. HRP2 is specific to Plasmodium falciparum, while pLDH is produced by all human-infecting Plasmodium species. [2]. The most commonly used RDTs feature a single test line that detects HRP2, followed by formats with two test lines, where the second line detects either pan-pLDH or Plasmodium vivax-specific pLDH [3]. Both HRP2 and pLDH lines can appear faint at low parasite densities; however, for a given parasite density, the pLDH line is typically less intense than the HRP2 line. Interestingly, HRP2 lines may also appear weak at very high parasite densities [4, 5]. More than 328 million RDTs were performed globally in 2023 [6]. The vast majority took place in the World Health Organization (WHO) African Region (266 million, 81%) and the South-East Asia Region (44 million, 14%). In Africa, RDTs are used nearly four times more often than microscopy for malaria diagnosis. Despite this heavy reliance on RDTs for confirming infection, many h (...truncated)