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Plasmodium ovale curtisi (Poc) and Plasmodium ovale wallikeri (Pow) represent distinct non-recombining Plasmodium species that are increasing in prevalence in sub-Saharan Africa. Though they circulate sympatrically, co-infection within human and mosquito hosts has rarely been described. Separate 18S rRNA real-time PCR assays that detect Poc and Pow were modified to allow species determination in parallel under identical cycling conditions. The lower limit of detection was 0.6 plasmid copies/μL (95% CI 0.4–1.6) for Poc and 4.5 plasmid copies/μL (95% CI 2.7–18) for Pow, or 0.1 and 0.8 parasites/μL, respectively, assuming 6 copies of 18s rRNA per genome. However, the assays showed cross-reactivity at concentrations greater than 103 plasmid copies/μL (roughly 200 parasites/μL). Mock mixtures were used to establish criteria for classifying mixed Poc/Pow infections that prevented false-positive detection while maintaining sensitive detection of the minority ovale species down to 100 copies/μL (<1 parasite/μL). When the modified real-time PCR assays were applied to field-collected blood samples from Tanzania and Cameroon, species identification by real-time PCR was concordant with nested PCR in 19 samples, but additionally detected two mixed Poc/Pow infections where nested PCR detected a single Po species. When real-time PCR was applied to oocyst-positive Anopheles midguts saved from mosquitoes fed on P. ovale-infected persons, mixed Poc/Pow infections were detected in 11/14 (79%). Based on these results, 8/9 P. ovale carriers transmitted both P. ovale species to mosquitoes, though both Po species could only be detected in the blood of two carriers. The described real-time PCR approach can be used to identify the natural occurrence of mixed Poc/Pow infections in human and mosquito hosts and reveals that such co-infections and co-transmission are likely more common than appreciated. Author summary: Plasmodium ovale, one of five species of malaria known to infect humans, in fact represents two distinct species, P. ovale curtisi (Poc) and wallikeri (Pow), that can only be distinguished using molecular diagnostics. Though Poc and Pow circulate in the same regions in Africa and Asia, mixed infections, where both are found in the same human host, have rarely been described. In this study, we modified existing real-time PCR assays targeting 18S rRNA and developed an algorithm to detect mixed Poc/Pow infections. We then applied these assays to field-collected samples from Tanzania and Cameroon, including blood samples from P. ovale-infected persons and P. ovale-positive mosquito midguts saved from mosquito feeding assays. We detected both Poc and Pow in roughly 10% of human P. ovale blood-stage infections, and surprisingly, in a majority of blood-fed mosquitoes. This suggests that Poc and Pow co-infect the same hosts more frequently than previously realized. [ABSTRACT FROM AUTHOR]