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Malaria rapid diagnostic tests (RDTs) are widely used to detect malaria parasites among patients who suspected malaria infections in malaria-endemic areas where microscopy is unavailable. Nevertheless, little is known about the performance of RDTs in detecting  Plasmodium  mixed infections. The present study aimed to evaluate the discordant results between RDTs and microscopy/polymerase chain reaction (PCR) in detecting  Plasmodium  mixed infections. The PubMed (MEDLINE), Web of Science, and Scopus databases were systematically reviewed to identify related studies that reported the performance of RDTs in detecting  Plasmodium  mixed infections .  Studies were grouped according to the different RDT types including RDT type 2 (pf-HRP2/pan-aldolase), RDT type 3 (pf-HRP2/pan-pLDH), RDT type 4 (Pf-LDH/pan-pLDH), RDT type 5 (Pf/Pv-pLDH), and RDT type 6 (pf-HRP2/Pv-pLDH) for subgroup analysis. The estimates of the different proportions in each analysis group that were visually summarized in a forest plot showed the odds ratio (OR) and 95% confidence interval (CI). Plots were drawn using RevMan (version 5.3; Cochrane Community). Twenty-eight studies were included in the present study. Overall, the meta-analysis showed that RDTs could detect a significantly higher proportion of  Plasmodium  mixed infections than microscopy (p = 0.0007, OR = 3.33, 95% CI 1.66–6.68). Subgroup analysis demonstrated that only RDTs targeting Pf-specific histidine-rich protein 2 (HRP2)/pan-specific lactate dehydrogenase (LDH) could detect a significantly higher proportion of  Plasmodium  mixed infections than microscopy (p = 0.004, OR = 8.46, 95% CI 2.75–26.1). The subgroup analysis between RDTs and PCR methods demonstrated that RDTs targeting Pf-specific HRP2/Pv-specific LDH could detect a significantly lower proportion of  Plasmodium  mixed infections than PCR methods (p = 0.0005, OR = 0.42, 95% CI 0.26–0.68). This is the first study to summarize the discordant results between RDTs and microscopy/PCR in detecting  Plasmodium  mixed infections. Malaria RDTs targeting Pf-HRP2/pan-pLDH could detect a higher proportion of  Plasmodium  mixed infections than microscopy, while RDTs targeting Pf-HRP2/Pv-specific LDH could detect a lower proportion of  Plasmodium  mixed infections than PCR methods. The results of this study will support the selection and careful interpretations of RDTs for a better diagnosis of  Plasmodium  mixed-species infections and appropriate treatment of malaria patients in endemic and non-endemic settings.

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Summary of discordant results between rapid diagnosis tests, microscopy, and polymerase chain reaction for detecting Plasmodium mixed infection: a systematic review and meta-analysis